Tissue-derived tumor growth inhibitors, methods of preparation and uses thereof.

Description: TECHNICAL FIELD [Detailed description of the invention]

Background of the invention Bichel [Bichel, Nature 231; 449-450 (1971)] reports that excision of many tumors from ascites tumor-carrying mice during the tumor growth equilibrium period results in significant growth of the remaining tumor cells. When acellular ascites obtained from mice carrying a fully advanced ascites tumor was injected into mice having a proliferative ascites tumor, the proliferation of ascites cells was significantly suppressed. Bichel (ibid.) Surgically combined two mice, one with advanced tumors and one with early tumors, in [parabiotic] mice, where early tumor growth was significantly suppressed. Also knows. Based on these findings, a diffusible inhibitory present in acellular ascites that circulates in the peritoneum of cooperatoid mice and is produced by well-advanced ascites tumors. The existence of the principle) was assumed [Bichel, Europ.J.Cancer 6: 291-296 (1970) and Bichel (ibid.)]. Although the nature of this suppressor has not been clarified, it was speculated that the growth rate of ascites tumors depends on the amount of tumor tissue and is determined by the amount of suppressor produced. Substances with tumor growth inhibitory activity are also described [Holley et al., Proc. Natl. Acad. Sci. USA 77:5989 (1980) and Holley et al., Cell. Biol.Int, Reports 7: 525-526 (1983)]. These publications report that growth inhibitors isolated from African green monkey BSC-1 cells suppressed the growth of BSC-1 cells, human breast cancer cells and normal human milk cells. Recently, this substance is the same as or very close to the 25,000 dalton double-stranded human platelet-derived polypeptide called B-TGF [Assoian et al., J. Biol. Chem. 258: 7155-7160 (1983)]. [Tucker et al., Science 226: 705-707 (1984); Roberts et al., Proc. Nat. Acad. Sci. 82 (Jan): 119-123 (1985)]. Mc Mahon et al. Also extracted 26000 daltons of a substance that suppresses the growth of non-malignant rat hepatocytes from the rat liver but not the growth of malignant rat hepatocytes [Proc. Natl. Acad. Sci. USA 79,456-460. (1982)]. Other growth inhibitors were identified in cultured chick spinal cord cells [Kagen et al., Experimental Neurology 58: 347-360 (1970); Harriston et al., Proc. Natl. Acad. Sci. USA 77: 423-427 (1980) and Steck et al., J. Cell Biol. 83: 562-575 (1979)]. Iwata et al. [J. Cellular Biochem. Suppl. 5: 401 (1982)] describe a microtiter plate system for testing growth stimulating and growth inhibitory activity. Todaro et al. [Todaro et al., Tumor Cell Heterogeneity; Origins and Implications, Bristol-Myers Cancer Symposia, Volume4, Owens, AH, Coffee, DS and Baylin, SB (Academic) Press, 1982), pp.205-224] and Iwata et al. [Fed.Proc.Fed.AM.Soc.Exp.Biol.42:1833 (1983)] It was reported that the tumor suppressive activity was isolated from. The findings contained in these reports are preliminary and not detailed. A patent application involving Kenenth K. Iwata as a co-inventor filed with the U.S. Patent and Trademark Office on April 20, 1984 (US Serial NNo.602,520) (Invention title Substantially Purified Tumor Growth Inhibitory Factor (TIF)) ") Relates to the preliminary identification of less specific substances present and derived from human tumor cells grown during culture. The material has already been reported [Todaro et al., Tumor Cell Heterogeneity; Origins and Implications, Bristol-Myers Cancer Symposia, Vol.4, Owens, AH, DS, and Baylin, SB (Academic). Press, 1982), pp.205-224; Iwata et al., Fed.Proc.Fed.Am, Soc.Exp.Biol.42:1833 (1983)] Similar to tumor suppressor activity. Todaro [Todaro, GJ, Epigenetic Regulation of Cancer, Terry Fox Cancer Research Conference (Vancouver, Canada, Columbia College, Vancouver, Canada) Abs.13 (1984)] recently has a tumor cell growth inhibitory effect on 70 and 90 tumor cells, respectively. We reported two factors composed of amino acid residues. No disclosure was made regarding the source of these factors, i.e. the cell, tissue type of species or method of purification of the factors. Abstract of the invention The present invention relates to an acidified ethanol extract derived from a human tissue composed of a plurality of acidic polypeptides, each of which has a molecular weight of less than about 20000 daltons and allows normal human foreskin fibroblast growth. It has the property of suppressing the growth of human tumor cells and established mink lung cell line (CCL64) while stimulating. The inhibitory activity on human tumor cell growth was not compromised by raising the temperature of the acidified ethanol extract to about 100 ° C for about 3 minutes or by adding acetic acid, at about 4 ° C rather than about 23 ° C. The inhibitory activity is higher when the acidified ethanol extract is prepared. The present invention also relates to an acidified ethanol extract derived from a human umbilical cord that has been treated to remove substantially all blood, all extracellular soluble components and substantially all intracellular soluble components. The extract has an apparent molecular weight of less than about 30,000 daltons under non-reducing conditions and proliferates human tumor cells and established mink lung cell lines (CCL64) while stimulating the growth of normal human foreskin fibroblasts. Has the property of suppressing. The inhibitory activity on human tumor cell growth is broken even if the temperature of the acidified ethanol extract is raised to about 100 ° C for about 3 minutes, or if the acidified ethanol extract is added with acetic acid up to about 1.0 mol in acetic acid. Absent. The present invention also relates to a method for producing an acidified ethanol extract derived from a human tissue consisting of a plurality of acidic polypeptides such as human umbilical cord or human placenta, each of which has a molecular weight of less than about 20000 daltons. It has the property of suppressing the growth of human tumor cells and established mink lung cell lineage (CCL64) while stimulating the growth of normal human placental umbilical cord. The method is lysed by treating the tissue under suitable conditions. It produces cell and cell-derived solubilized proteins, recovers the lysed proteins, separately recovers polypeptides with an apparent molecular weight of less than about 20000 daltons from the lysed proteins, and validates the recovered polypeptides in humans. A polypeptide that suppresses the growth of tumor cells, suppresses the growth of established mink lung cell lineage (CCL64), or promotes the growth of normal human capsule fibroblasts has been identified, and the identified polypeptide has been identified. Consists of recovering the containing oxidizing ethanol extract. The present invention also has an apparent molecular weight of approximately 20,000 to 30,000 daltons and has the property of suppressing the growth of human tumor cells and established mink lung cell lines (CCL64) while stimulating the growth of normal human foreskin fibroblasts. Tissue-derived growth The present invention relates to a method for producing an acidified ethanol extract from a human umbilical cord consisting of inhibitor (TGI). The method removes veins and arteries from the umbilical cord tissue, cleans the tissue to remove all trace blood, treats the tissue to produce lysed cells, removes soluble proteins derived from the cells, under suitable conditions. The remaining protein was lysed and isolated from the degraded cells by acidified ethanol extraction to produce the lysed protein, and TGIs with an apparent molecular weight of approximately 20,000 to 30,000 daltons were separately recovered from the lysed proteins and thus separately recovered. TGI was tested to identify the activity of suppressing the growth of human tumor cells, suppressing the growth of established mink lung cell lineage (CCL64) and promoting the growth of normal human foreskin fibrilblasts, and thus identified TGIs. It consists of recovering the acidified enol extract containing. The present invention also has an apparent molecular weight of approximately 5000-16000 daltons and is a property that suppresses the growth of human tumor cells and established mink lung cell lines (CCL64) but not the growth of normal human foreskin fibroblasts. Provided is a substance called a tissue-derived growth inhibitor-1 (TGI-1), which comprises at least one polypeptide having. The substance is a) When an acidified ethanol extract is applied to a high performance liquid chromatograph using a linear gradient of acetonitrile containing 0.05% trifluoroacetic acid in about 26-36% acetonitrile, it can be recovered as the specified activity, about 17 Ethanol extracts acidified using a linear gradient of -23% 2-propanol and 0.05% trifluoroacetic acid containing 2-propanol can be recovered as defined activity when subjected to high performance liquid chromatography. b) When subjected to high performance liquid chromatography using a linear gradient of 2-propanol containing 0.05% trifluoroacetic acid, it is eluted with about 26% 2-ethanol and preferentially suppresses the growth of human tumor cells, but established mink. Activity that does not suppress the growth of lung cell line (CCL64) and activity that suppresses the growth of established minced lung cell line (CCL64) preferentially by elution with about 23% 2-ethanol but does not suppress the growth of human tumor cells 2 Can be separated into individual activities. The present invention also provides a method for producing TGI-1. The present invention also has an apparent molecular weight of approximately 20,000 to 30,000 daltons and does not suppress the growth of normal human foreskin fibroblasts but suppresses the growth of human tumor cells and established mink lung cell lines (CCL64). Provided is a substance consisting of at least one polypeptide having a substance called a tissue-derived growth inhibitor (TGI). The material is recoverable as defined activity when the acidified ethanol extract is subjected to high performance liquid chromatography using a linear gradient of acetonitrile containing 0.05% trifluoroacetic acid in about 28-34% acetonitrile. About 0.6-0.7M It can be eluted as a single peak with defined activity when applied to a cation exchange resin with NaCl using a linear gradient of NaCl. The present invention also has an apparent molecular weight of approximately 5000-16000 daltons and does not suppress the growth of normal human foreskin fibroblasts but suppresses the growth of human tumor cells and established mink lung cell lines (CCL64). Provided is a substance called tissue-derived growth inhibitor-2 (TGI-2), which is composed of at least one polypeptide having. The substance is recoverable as defined activity when the acidified ethanol extract is subjected to high performance liquid chromatography using a linear gradient of acetonitrile containing 0.05% trifluoroacetic acid in about 35-39% acetonitrile. An ethanol extract acidified using a linear gradient of 2-propanol containing about 23-27% 2-propanol and 0.05% trifluoroacetic acid can be recovered as defined activity when subjected to high performance liquid chromatography. .. The present invention also provides a method for producing TGI-2. Furthermore, the present invention provides a heterogeneous population of polypeptides called CM-I, CM-II, CM-III and CM-IV. Each of the above-mentioned polypeptides has a property of substantially suppressing the growth of human tumor cells, and has an effect of substantially suppressing the growth of the established mink lung cell line (CCL64) except for CM-I. Peptides also do not suppress the growth of normal human foreskin fibroblasts. The aggregate can be recovered by subjecting the acidified ethanol extract to a cation exchange chromatograph. The present invention also provides a method for producing a heterogeneous aggregate of polypeptides called CM-I, CM-II, CM-III and CM-IV. Drugs containing an effective amount of extracts TGI-1, TGI, TGI-2 or heterogeneous aggregates of polypeptides called CM-I, CM-II, CM-III and CM-IV and suitable pharmaceutical carriers. A composition is provided, and a method for suppressing the growth of human tumor cells by contacting the composition with cells is provided. The composition is also used for burn ulcers and wound healing. The present invention also relates to a method of detecting the presence of a tumor. The method is the amount of TGI-1, TGI or TGI-2 present in the sample sample or the amount of heterogeneous aggregates of polypeptides called CM-I, CM-II, CM-III or CM-IV. Is quantitatively measured and the measured amount thus obtained is compared with the amount present in a normal sample sample, and a significant difference in the amount indicates the presence of a tumor. Furthermore, the present invention presents the amount of transforming and proliferating factor alpha (TGF-α) present in the sample sample and the amount of TGI-1, TGI or TGI-2 or CM-I, CM-II, CM-III or CM. The amount of heterogeneous aggregate of polypeptide called -IV is measured separately and quantitatively, and TGI-1, TGI, TGI-2 or heterogeneous assembly in the sample with respect to the amount of TGF-α that is impaired. It consists of measuring the ratio of body mass, measuring the control ratio of normal sample samples, and comparing the ratio of normal samples with the ratio of samples, and a significant change in the ratio suggests the presence of a tumor. It also relates to a method for detecting a tumor. Finally, the present invention refers to the presence of one or more TGI-1, TGI, TGI-2, CM-I, CM-II, CM-III and CM-IV in a sample from a tumor-bearing sample. The present invention relates to a method for determining a tumor type in which the presence or absence of a specific combination thereof, or the presence of a specific amount or a relative amount thereof is an index of a specific tumor type. The drawings will be described below. Fig. 1 Gel hyperchromatography (23 ° C) FIG. 1 shows the elution pattern of gel hyperchromatography at 23 ° C of the crude acidic ethanol extract of the human umbilical cord. 2 g of acidic ethanol extract in 1.50 ml of 1.0 M acetic acid was applied to a 14 × 100 cm column (Amicon; # 86012) containing Bio-Gel P10 and eluted at a flow rate of 7 ml / min. Super rack equipped with C-type collection rack (LKB) One flux was collected by Rac (LKB2211). 1 ml was taken from each flux (1 / each flux) and transferred to a 12 × 75 mm sterile snaptotoptieuve (Falcon 2058). TGI activity was measured by the method described in the Substances and Methods section. The inhibitory effect of A549 human lung cancer cells is indicated by a white triangle, and that of mink lung cells (CCL64) is indicated by a white circle. Absorption (-) at 280 nm was measured using a Uvicood S (LKB2138) with a maximum absorption scale range of 1.0 AUFS and a 1 channel chart recorder (LKB2210) (chat speed 1 mm / min). Fig. 2 Gel hyperchromatography (4 ° C) Figure 2 shows the elution pattern of gel hyperchromatography at 4 ° C of the crudely acidic ethanol extract of the human umbilical cord. 2 g of acidic ethanol extract in 150 ml of 1.0 M acetic acid was applied to a 14 × 100 cm column (Amicon; # 86012) containing Bio-Gel P10 and eluted at a flow rate of 7 ml / min. Super rack equipped with C-type collection rack (LKB) One fraction was collected by Rac) (LKB2211). 1 ml was taken from each flux (1 / each flux) and transferred to a 12 × 75 mm sterile snaptotoptieuve (Falcon 1058). TGI activity was measured by the method described in the Substances and Methods section. The inhibitory effect of A549 human lung cancer cells is indicated by a white triangle, and that of mink lung cells (CCL64) is indicated by a white circle. Absorption (-) at 280 nm was measured using a Uvicord S (LKB2138) with a maximum absorption scale range of 1.0 AUFS and a 1 channel chart recorder (LKB2210) (chart speed 1 mm / min). Fig. 3 Inhibition of cell proliferation and stimulation of normal human cells by fractionation of gel hyperchromatography (4 ° C) Figure 3 shows the elution pattern of gel hyperchromatography at 4 ° C of the crudely oxidizing ethanol extract of the human umbilical cord. The acidic ethanol extract in 150 ml of 1.0 M acetic acid was applied to a 14 × 100 cm column (Amicon; # 86012) containing Bio-Gel P10 and eluted at a flow rate of 7 ml / min. One flux was collected by Super Rac (LKB2211) equipped with C-type collection rack (LKB). 1 ml was taken from each flux (1 / each flux) and transferred to a 12 × 75 mm sterile snaptotoptieuve (Falcon 2058). TGI activity was measured by the method described in the Substances and Methods section. The inhibitory effect of A549 human lung cancer cells is indicated by a white triangle, and that of mink lung cells (CCL64) is indicated by a white circle. The irritation of normal human human fibroblast is indicated by a white square. Absorption (-) at 280 nm was measured using a Uvicord S (LKB2138) with a maximum absorption scale range of 1.0 AUFS and a 1 channel chart recorder (LKB2210) (chart speed 1 mm / min). Fig. 4 Reverse-Phase High Performance Liquid Chromatography of Active Fractions from Gel Hyperchromatography (HPLC) Fraxion 4 in a gel hyperchromatography with bio-gel P10 of acidified ethanol extract of human umbilical cord (protein 65.8 mg) was lyophilized and resuspended in 10 ml of 0.05% trifluoroacetic acid (TFA). It was. Fraction 4 was the first flux after the main peak of absorption at 280 nm (see Figure 2). This sample was placed in a Beckman TJ-6 centrifuge and centrifuged at 3000 rpm for 20 minutes to remove insoluble material. The above supernatant (supernatant) was injected in three portions using a 2 ml Water's U6K injection device with a sample loop. Then sample the u BONPAPAK C₁₈It was hung on a column (0.78 x 30 cm) (Waters # 84176). The flow rate was 2 ml / min, and elution was monitored at 206 nm (-) with a sensitivity of 2.0 AUFS using a Waters UV measuring instrument (Waters Model 481). A linear concentration gradient from 0.25% of 0.05% TFA-containing acetonitrile (30 minutes), followed by a 25-45% linear concentration gradient of 0.05% TFA-containing acetonitrile (240 minutes), and a 45-100% linear concentration of 0.05% TFA-containing acetonitrile. Elution was performed by gradient (30 minutes). It was collected in 12 ml fractions using Super Rack (LKB2211). 1 ml from each fraction was transferred to polystyrene tube (Falcon 2058) (12 x 75 mm) containing 50 μg bovine serum albumin (sigma B) and 50 μl 1.0 M acetic acid, and TGI activity was subjected to as described in Substances and Methods. A549 human lung cancer cell inhibition is indicated by a white triangle and that of mink lung cells (CCL64) is indicated by a white circle. The solvent gradient is indicated by the dotted line (-----). Fig. 5 Rechromatography by HPLC of TGI active pool (TGI-1) from high performance liquid chromatograph (HPLC) In the HPLC chromatograph of Fig. 4, TGI-active pool fraction (1.5 mg) (fluxion 13-22) eluted with 28-34% acetonitrile was lyophilized and 0.05% trifluoroacetic acid (TFA). ) Resuspended in 2 ml. This sample was placed in a Beckman table top centrifuge (Backman TJ-6) and centrifuged at 3000 rpm for 20 minutes to remove insoluble substances. The above supernatant (supernatant) was injected in two portions using a 2 ml Water's U6K injection device with a sample loop. Then sample the u BONPAPAK C₁₈It was hung on a column (0.39 x 30 cm) (Waters # 27324). The flow rate was 1 ml / min, and elution was monitored at 206 nm (-) with a sensitivity of 2.0 AUFS using a Waters UV measuring instrument (Waters Model 481). Elution was performed with a 0-15% linear concentration gradient of 2-propanol containing 0.05% TFA (20 minutes), followed by a 15-35% linear concentration gradient of 2-propanol containing 0.05% TFA (120 minutes). It was collected in 4 ml fractions using Super Rack (LKB2211). 1 ml of each fraction was transferred to polystyrene tube (Falcon 2058) (12 x 75 mm) containing 50 μg of bovine serum albumin (Sigma A-6003) and 50 μg of 1.0 M acetic acid, and TGI activity was assessed as described in Substances and Methods. A549 human lung cancer cell inhibition is indicated by a white triangle, and that of mink lung cell (CCL64) is indicated by a white circle. The solvent gradient is indicated by the dotted line (----). Fig. 6 Rechromatography by reverse phase HPLC of TGI activity pool (TGI-2) from high performance liquid chromatography (HPLC) In the HPLC chromatograph of Fig. 4, TGI-active pool fractions (0.8 mg) (fluxions 25-31) eluted with 35-39% acetonitrile were lyophilized and 0.05% trifluoroacetic acid (TFA). ) Resuspended in 2 ml. This sample was placed in a Beckman TJ-6 centrifuge and centrifuged at 3000 rpm for 30 minutes to remove insoluble material. The above supernatant (supernatant) was injected in two portions using a 2 ml Water's U6K injection device with a sample loop. Then sample the u BONPAPAK C₁₈It was hung on a column (0.39 x 30 cm) (Waters # 27324). The flow rate was 1 ml / min, and elution was monitored at 206 nm (-) with a sensitivity of 1.0 AUFS using a Waters UV measuring instrument (Waters Model 481). Elution was performed with a linear concentration gradient from 0.15% of 2-propanol containing 0.05% TFA (20 minutes), followed by a 15-35% linear concentration gradient of 2-propanol containing 0.05% TFA (120 minutes). It was collected in 4 ml fractions using Super Rack (LKB2211). 1 ml from each fraction was transferred to polystyrene tube (Falcon 2058) (12 x 75 mm) containing 50 μg bovine serum albumin (Sigma A-6003) and 50 μg 1.0 M acetic acid, and the TGI activity was assessed as described in Substances and Methods. A549 human lung cancer cell inhibition is indicated by a white triangle and that of mink lung cells (CCL64) is indicated by a white circle. The solvent gradient is indicated by the dotted line (----). Fig. 7 Reverse-Phase High Performance Liquid Chromatography of Active Fractions from Gel Hyperchromatography (HPLC) Fraction 5 in gel hyperchromatography with Bio-Gel P10 of acidified ethanol extract of human umbilical cord was lyophilized and resuspended in 4 ml of 0.05% trifluoroacetic acid (TFA). Fraction 5 was the second flux after the main peak of absorption at 280 nm (see Figure 2). This sample was placed in a Beckman TJ-6 centrifuge and centrifuged at 3000 rpm for 20 minutes to remove insoluble material. The above supernatant (supernatant) was injected in two portions using a 2 ml Water's U6K injection device with a sample loop. Next, the sample (1.3 ml total volume) is added to u BONDAPAK C.₁₈It was hung on a column (0.78 x 30 cm) (Waters # 84176). The flow rate was 2 ml / min, and elution was monitored at 206 nm (-) with a sensitivity of 2.0 AUFS using a Waters UV measuring instrument (Waters Model 481). A linear concentration gradient from 0.25% of 0.05% TFA-containing acetonitrile (30 minutes), followed by a 25-45% linear concentration gradient of 0.05% TFA-containing acetonitrile (240 minutes), and a 45-100% linear concentration of 0.05% TFA-containing acetonitrile. Elution was performed by gradient (30 minutes). It was collected in 12 ml fractions using Super Rack (LKB2211). 1 ml of each fraction was transferred to polystyrene tube (Falcon 2058) (12 x 87 mm) containing 50 μg of bovine serum albumin (sigma A6003) and 50 μg of 1.0 M acetic acid, and TGI activity was assessed as described in Substances and Methods. A549 human lung cancer cell inhibition is indicated by a white triangle, and that of mink lung cell (CCL64) is indicated by a white circle. The solvent gradient is indicated by the dotted line (----). Fig. 8 Reverse Phase HPLC Rechromatography of TGI Active Pool (TGI-1) from High Performance Liquid Chromatograph (HPLC) TGI-active pool flux (1.1 mg) (fluxion 14-25) eluted with 29-34% acetonitrile in Figure 7 was lyophilized and resuspended in 2 ml of 0.05% trifluoroacetic acid (TFA). I let you. This sample was placed in a Beckman TJ-6 centrifuge and centrifuged at 3000 rpm for 20 minutes to remove insoluble material. The above supernatant (1.6 ml) (supernatant) was injected in two portions using a 2 ml Water's U6K injection device with a sample loop. Then sample the u BONDAPAK C₁₈It was hung on a column (0.78 x 30 cm) (Waters # 84174). The flow rate was 1 ml / min, and elution was monitored at 206 nm (-) with a sensitivity of 1.0 AUFS using a Waters UV measuring instrument (Waters Model 481). 0-10% linear concentration gradient of 2-propanol containing 0.05% TFA (20 minutes), then 10-35% linear concentration gradient of 2-propanol containing 0.05% TFA (220 minutes), and 2-propanol containing 0.05% TFA. Elution was performed with a 35-45% linear concentration gradient (20 minutes) and a 45-100% linear concentration gradient of 2-propanol containing 0.05% TFA (20 minutes). It was collected in 8 ml fractions using Super Rack (LKB2211). 1 ml of each fraction was transferred to polystyrene tube (Falcon 2058) (12 x 75 mm) containing 50 μg of bovine serum albumin (sigma 6003) and 50 μg of 1.0 M acetic acid, and the TGI activity was agitated as described above. A549 human lung cancer cell inhibition is indicated by a white triangle, and that of mink lung cell (CCL64) is indicated by a white circle. The solvent gradient is indicated by the dotted line (----). Fig. 9 Cation exchange chromatograph of human umbilical cord extract CM-TRISACRYL equivalent 0.1M ammonium acetate, pH 4.0 (1.0M) Resuspended in (containing NaCl). The resin was equilibrated for 3 hours and degassed at 4 ° C. 20 ml of this resin was packed in a 1.6 × 40 cm column (Pharmacia; # 19-0362-01) and washed with 2 column volumes of 1.0 M ammonium acetate, pH 4.0 and then 0.01 M ammonium acetate. The column was washed until the conductivity and pH of the eluate were equal to those of the equilibrated buffer (0.01 M ammonium acetate, pH 4.0). 1 g of acidified ethanol extract of the human umbilical cord was resuspended in 50 ml of 1.0 M acetic acid and dialyzed against column equilibrated buffalo at 4 ° C until pH and conductivity were equal to those of buffalo. This dialyzed acidified ethanol extract was applied to the column at a flow of 1 ml / min at 4 ° C, and the column was washed with an equilibrated buffer until the absorption (-) at 280 nm reached the lowest value (absorption is Ubicode S). (LKB2138), monitored with a sensitivity of 1.0 AUFS). Next, a gradient mixer (Falmacia GM-1, # 19-0495-01) was used to apply a rising linear molar concentration gradient (200 ml) of 0.01-1.0 M ammonium acetate and pH 4.0. At the end of the gradient, an additional 30 ml of 1.0 M ammonium acetate, pH 4.0 was run through the column. Two ml of each flux was collected in a 12 x 100 mm polystyrene tube (Columbia Diagnostics B-2564) using a Super Luxe Fraction Collector (LKB2211). From each flux, 1 ml was transferred to a 12 × 75 mm tube (Falcon 2058) containing 50 μg bovine serum albumin (sigma A6003) and 50 μl 1.0 M acetic acid. This was lyophilized and the TGI activity was evaluated by the method described in the column of substance and method. A549 The inhibitory effect of human lung cancer cells is indicated by a white triangle, and the inhibitory effect of mink lung cells (CCL64) is indicated by a white circle. The salt gradient is indicated by the dotted line (---). Fig. 10 Rechromatography of fractions pooled from cation exchange chromatograph CM-TRISACRYL was prepared as described in the description of Figure 9. Materials from fractions containing CM III and CM IV are pooled, lyophilized and resuspended in 50 ml of 1.0 M acetic acid until the pH and conductivity are equal to those of the column equilibrated buffer 4 Dialysis was performed at ° C. The sample was run on a column at 4 ° C. at a flow rate of 1 ml / min and the column was washed with 120 ml of equilibrated buffer. Ubicode S (LKB2138), absorption (-) at 280 nm was monitored with a sensitivity of 1.0 AUFS. Next, a gradient mixer (Falmacia GM-1, # 19-0495-01) was used to apply a rising linear molar concentration gradient (100 ml) of 0.01-1.0 M ammonium acetate and pH 4.0. At the end of the gradient, an additional 30 ml of 1.0 M ammonium acetate, pH 4.0 was run through the column. Two ml of each flux was collected in a 12 x 100 mm polystyrene tube (Columbia Diagnostakes B-2564) using a Super Luxe Fraction Collector (LKB2211). From each fraction, 1 ml was transferred to a 12 × 75 mm tube (Falcon 2058) containing 50 μg bovine serum albumin (sigma A6003) and 50 μl 1.0 M acetic acid. This was lyophilized and the TGI activity was evaluated by the method described in the column of substance and method. A549 Inhibition of human lung cancer cells is indicated by a white triangle, and inhibition of mink lung cells (CCL64) is indicated by a white circle. The salt gradient is indicated by the dotted line (---). Fig. 11 Separation of TGI by cation exchange chromatograph (4 ° C) 1.65 mg of protein extract prepared as described in Part 2 of the experiment was thoroughly dialyzed against 20 mM ammonium acetate (pH 4.5) and pre-equilibriumed with 20 mM ammonium acetate (pH 4.5). The CM-TRISACRYL was placed on a 5 ml column (1 × 6.3 cm) and collected in 1.65 ml fractions (12 × 100 mm polystyrene protein). After sample addition, 20 mM the column until the absorption at 280 nm measured by the Bausch and Lomb 1001 spectrophotometer using a 1 cm optical path quartz substrate returns to the baseline value (less than 0.003). Washed with ammonium acetate pH 4.5. A linear salt gradient (20 mM ammonium acetate, 0-1.0 M NaCl in pH 4.5) was applied and the absorption of 1.65 ml of each fraction was measured at 280 nm as described above. From each fraction, 10 μm was transferred to a 12 × 75 mm tube containing 50 μg bovine serum albumin (sigma A6003) and 1.0 M acetic acid 50 μm, lyophilized and transferred to A549 human lung cancer cells according to the method described in the Substances and Methods section. Inhibitory activity against ( ... ) was performed. The NaCl gradient (---) was measured in the conductivity of a suitable sample diluted 100-fold with water (YSI model 32 conductivity meter). Fig. 12 Separation of TGI by anion exchange chromatograph (4 ° C) As described in Part 2 of the experiment, 1.65 mg of the prepared protein was thoroughly dialyzed against 20 mM Tris-HCl (pH 8.0) and centrifuged at 3,000 g for 15 minutes to make it clear. DEAE-TRISACRYL was prepared by first suspending the resin in 20 mM Tris-HCl (pH 8.0) containing 1.0 M NaCl for 3 hours and then in 0.5 M Tris-HCl (pH 8.0) for 1 hour. The precipitated resin was washed on a Büchner funnel with 1000 ml of water, finally resuspended in 20 mM Tris-HCl (pH 8.0), degassed and poured into a 5 ml column (1 x 6.3 cm) to pour the resin into 20 mM Tris. -Balanced with HCl (pH 8.0). The transparent sample was placed on a column, and the absorption (-) at 280 nm, the inhibitory activity on mink lung cells ( ... ) and the NaCl gradient (---) were shown in Fig. 11 and the substances and methods. Measured as described. The NaCl gradient was carried out in the range of 0 to 1.0 M NaCl in 20 mM Tirs-HCl (pH 8.0). Fig. 13 Separation of TGI by cation exchange chromatograph (4 ° C) CM-TRISACRYL was prepared as described in the description of Figure 9, except that the final equilibration buffer was 20 mM ammonium acetate, pH 4.5. The protein extract (9.9 mg) prepared as described above was thoroughly dialyzed against 20 mM ammonium acetate (pH 4.5) and a 15 ml column (1.5 ×) of CM-TRISACRYL in 20 mM ammonium acetate (pH 4.5). 8.5 cm). Absorption (-) at 280 nm and inhibitory activity on A549 human lung cancer cells ( ... ... ) were measured as described in the description of FIG. The volume of 0-1.0 M linear gradient of NaCl was 150 ml and the volume of fraction was 3.7 ml. Figure 14 Reverse-Phase High Performance Liquid Chromatographs of Active Fractions from Cation Exchange Chromatographies (HPLC) Fractions 59-78 of cation exchange chromatographies (see Figure 13) using CM-TRIS ACRYL in the human umbilical cord were collected, lyophilized and resuspended in 10 ml of 0.05% trifluoroacetic acid (TFA). I let you. A total of 20% of the dialysate containing 240 μg of protein was injected in 3 divided doses using a 2 ml Water's U6K injection device with a sample loop. Then the sample is uBONDAPAKC₁₈It was hung on a column (0.39 x 30 cm) (Waters 27324). The flow rate was 1 ml / min, and elution was monitored at 206 nm (-) with a sensitivity of 0.5 AUFS using a Waters UV measuring instrument (Waters Model 481). 0-25% increasing linear concentration gradient of 0.05% TFA-containing acetonitrile (5 minutes), then 25-45% linear concentration gradient of 0.05% TFA-containing acetonitrile (15 minutes), and 45-80% of 0.05% TFA-containing acetonitrile Elution was performed with a linear concentration gradient (15 minutes) and finally with a 0.05% TFA-containing acetonitrile 80-100% linear concentration gradient (5 minutes). It was collected in 1 ml fractions using Super Rack (LKB2211). From each flux, 500 μm was transferred to polystyrene tube (Falcon 2058) (12 × 75 mm) containing 50 μg of bovine serum albumin (Sigma A0281) and 50 μg of 1.0 M acetic acid, and TGI activity was assessed as described in Substances and Methods. A549 human lung cancer cell inhibition is indicated by a white triangle, and that of mink lung cell (CCL64) is indicated by a white circle. The solvent gradient is indicated by the dotted line (----). Detailed description of the invention An acidified ethanol extract from human tissue containing multiple polypeptides was prepared. Each polypeptide of this extract has a molecular weight of less than about 20,000 daltons, each of which enhances the growth of normal human foreskin fibroblasts but inhibits the growth of human tumor cells and the established mink lung cell line (CCL64). Has characteristics. Heating the acidified ethanol extract to about 100 ° C. and maintaining it for about 3 minutes, or adding acetic acid up to about 1.0 molar concentration to the extract does not destroy the inhibitory activity of human tumor cell growth. Preparation of the acidified ethanol extract at about 4 ° C instead of about 23 ° C enhances the inhibitory activity of the extract. According to a preferred embodiment, the human tissue is the human umbilical cord, but it is of course possible to use another tissue such as the human placenta. Also, an acidified ethanol extract containing at least one acidic polypeptide was prepared from the human umbilical cord treated to remove substantially all blood, all extracellular soluble components and all intracellular soluble components. This polypeptide exhibits an apparent molecular weight of less than about 30,000 daltons under non-reducing conditions and promotes the growth of normal human foreskin fibroblast but inhibits the growth of human tumor cells and the established mink lung cell line (CCL64). Has characteristics. Even if the acidified ethanol extract is heated to about 100 ° C and maintained for about 3 minutes, the inhibitory activity of human tumor cell growth is not destroyed even if acetic acid up to about 1.0 molar concentration is added to the acidified ethanol extract. Techniques known to those of skill in the art, such as high performance liquid chromatographies and cation exchange chromatographies, may be used to prepare the various components of the acidified ethanol extract. As a result, a substance containing at least one polypeptide having an apparent molecular weight of about 5,000 to 16,000 daltons, which has a growth inhibitory effect on human tumor cells and the established mink lung cell line (CCL64) and does not inhibit the growth of human foreskin fibroblast, is prepared. Will be done. This substance is designated as a tissue-derived growth inhibitor (TGI-1) and can be recovered by high performance liquid chromatography of acidified ethanol extract. That is, ethanol acidified with a linear gradient of about 26-34% acetonitrile of acetonitrile containing 0.05% trifluoroacetic acid and about 17-23% 2-propanol linear gradient of 2-propanol containing 0.05% trifluoroacetic acid. When the extract is subjected to high performance liquid chromatography, it can be recovered as a predetermined activity. When TGI-1 is further eluted with a high performance liquid chromatographie with a 2-propanol linear gradient containing 0.05% trifluoroacetic acid, it is split into two active components. One activity is eluted with about 26% 2-propanol and preferentially inhibits the growth of human tumor cells but does not inhibit the growth of the established mink lung cell line (CCL64). Another activity is eluted with about 23% 2-propanol and preferentially inhibits the growth of established mink lung cell lines (CCL64) but does not inhibit the growth of human tumor cells. Also referred to as a tissue-derived growth inhibitor (TGI), which consists of at least one polypeptide, which also does not inhibit the growth of normal human foreskin fibroblasts, but human tumor cells and established mink lung cell lines (CCL64). Another substance with growth inhibitory activity against and is prepared. The apparent molecular weight of the TGI ranges from about 20,000 to 30,000 daltons and is defined from the acidified ethanol extract by a high performance liquid kramatgrafie with a linear gradient of about 28 to 34% acetonitrile of acetonitrile containing 0.05% trifluoroacetic acid. It is recovered as an activity, and when a NaCl linear gradient of about 0.6 to 0.7 M NaCl is eluted, it is recovered as a single peak predetermined activity from the CM-Tris acrylic (TRYSACRYL) resin after the cation exchange resin. Furthermore, a substance designated as tissue-derived growth inhibitor-2 (TGI-2) is obtained. This substance contains one or more polypeptides having an apparent molecular weight of about 5,000 to 16,000 daltons, which has growth inhibitory properties against human tumor cells and established mink lung cell lineage (CCL64) but does not inhibit the growth of normal human foramen fibroblast. It is recovered as a predetermined activity from the acidified ethanol extract by high performance liquid chromatography with about 35-39% acetonitrile linear gradient of acetonitrile containing 0.05% trifluoroacetic acid, and 0.05% trifluoroacetic acid is also obtained. Approximately 23-27% of 2-propanol containing 2-propanol recovered as a given activity from the acidified ethanol extract by high performance liquid chromatography with a linear gradient. In addition, heterogeneous aggregates of polypeptides, namely CM-I, CM-II, CM-III and CM-IV, are recovered by cation exchange chromatographies of acidified ethanol extracts. Each population has the property of inhibiting the growth of human tumor cells, and all but CM-I have the property of substantially inhibiting the growth of the established mink lung cell line (CCL64), all of which are normal humans. Does not inhibit the growth of foreskin fibroblast. Heterogeneous aggregates of polypeptides designated TGI-1, TGI, TGI-2 or CM-I, CM-II, CM-III and CM-IV or various combinations thereof are used as drug compositions. obtain. The drug composition comprises an effective amount of a heterogeneous assembly of polypeptides designated TGI-1, TGI, TGI-2 or CM-I, CM-II, CM-III and CM-IV with a suitable drug carrier. Including. Effective amounts of various tumor growth inhibitors may be varied by methods known to those of skill in the art, depending on treatment instructions, the patient or the time of tumor development. Also known to those skilled in the art are suitable carriers such as saline or other aqueous solutions, gels, creams and the like. Heterogeneous aggregates of polypeptides designated TGI-1, TGI, TGI-2 or CM-I, CM-II, CM-III and CM-IV are found in human tumor cells such as carcinomas, melanomas or leukemia cells. Can be used to inhibit growth. For this purpose, a heterogeneous aggregate of polypeptides designated as TGI-1, TGI, TGI-2 or CM-I, CM-II, CM-III and CM-IV in an amount effective for growth inhibition is used as a cell. Make contact. Heterogeneous aggregates of polypeptides designated TGI-1, TGI, TGI-2 or CM-I, CM-II, CM-III and CM-IV are also used to assist in the treatment of burns or healing of wounds. To. To do this, an effective amount of a heterogeneous aggregate of polypeptides designated TGI-1, TGI, TGI-2 or CM-I, CM-II, CM-III and CM-IV and a suitable drug carrier are used. The drug composition contained is brought into contact with a burn or wound. According to the present invention, each polypeptide contains a plurality of polypeptides, each polypeptide has a molecular weight of less than about 20,000 daltons, and each polypeptide has a growth inhibitory property between a human tumor cell and an established mink lung cell line (CCL64) and is normal. Disclosed are methods of preparing acidified ethanol extracts from human tissues that enhance the growth of human foreskin peptides. In the method, under suitable conditions, the tissue is treated, for example by freezing-thawing or homogenization of the tissue to produce lytic cells and lytic proteins derived from lytic cells, the lytic protein is recovered and from the lytic protein. Separately, a polypeptide having an apparent molecular weight of less than about 20,000 daltons is separated and recovered, and the separated and recovered polypeptide is used to inhibit the growth of human tumor cells or the growth of the established mink lung cell lineage (CCL64) or normal human foreskin fibroblast. The polypeptide showing hyperproliferation of the protein is identified, and the oxidizing ethanol extract containing the polypeptide thus identified is recovered. According to a preferred embodiment of the method of the invention, the tissue treatment involves thawing the frozen tissue at about 4 ° C for a suitable time, eg, 6 hours, for a suitable acidic extraction containing ethanol at about 4 ° C. The tissue is suspended in a buffer, the tissue is homogenized for an appropriate period of time to form a homogeneous tissue, and the homogeneous tissue is stirred at about 4 ° C for an appropriate time, for example overnight, to generate lysed cells and cells. It consists of steps that solubilize the derived protein. According to the present invention, preferred acidic extractions are about 375 ml of 95 (volume / volume)% ethanol, 7.5 ml of concentrated hydrochloric acid, 33 mg of fluorinated phenylmethylsulfonyl (PMSF) and 1 ml of aprotinin (Sigma A6012,). It is a mixture of 0.9% NaCl and 0.9% benzyl alcohol (with 9.8 units / ml of trypsin inhibitory unit) in about 192 ml of 4 ° C distilled water. According to a preferred embodiment of the invention, recovery of the soluble protein separates the lysed protein from the lysed cells, for example by centrifugation, and raises the pH to about 5.0, for example by the addition of ammonium hydroxide, eg, The precipitate is removed by centrifugation, for example the protein is precipitated from the supernatant by the addition of alcohol, ether or both, for example by allowing the solution to stand at about 4 ° C for about 48 hours and centrifuging. The precipitate is removed from the solution, the organic phase is removed from the precipitate, the precipitate is dried, for example in a fume food, and the dried precipitate is redissolved in a suitable solvent such as 1M acetic acid, for example for dialysis. Therefore, it consists of a step to remove the low molecular weight solute from the solution. The present invention further develops tissue-derived growth with an apparent molecular weight of approximately 20,000 to 30,000 daltons, which inhibits the growth of human tumor cells and the established mink lung cell line (CCL64) and promotes the growth of normal human foreskin umbilical cord fibroblast. It discloses a method for preparing an acidified ethanol extract containing an inhibitor (TGI) from the human umbilical cord. In this method, under suitable conditions, veins and arteries are removed from the umbilical cord tissue, the tissue is washed to completely remove trace blood, the tissue is treated to produce lysed cells, and cell-derived soluble proteins. The residual protein was solubilized and separated by acidified ethanol extraction to produce a lysate protein, and TGI having an apparent molecular weight of about 20,000 to 30,000 daltons was separated and recovered from the lysed protein, and the separated and recovered TGI was tested. The TGI-containing acidic ethanol extract identified as described above was identified as an activity that inhibits the growth of human tumor cells, inhibits the growth of established minced lung cell lineage (CCL64), and promotes the growth of normal human foreskin fibroblast. To collect. According to a preferred embodiment of the invention, tissue treatment involves thawing frozen tissue at about 4 ° C for a suitable time, eg, 2 hours, and incising at about 4 ° C to remove veins and arteries. Then, the tissue is suspended in a suitable buffer at about 4 ° C, and the tissue is homogenized for a suitable period of time to form lysed cells and soluble protein. Soluble protein is formed from the lysed cells by centrifugation, for example. The cells consist of steps that produce lysed proteins by separating and stirring the lysed cells in an extraction buffer at about 4 ° C for an appropriate period of time, eg overnight. The preferred acid extraction buffers of the present invention are approximately 375 ml 95 (volume / volume)% ethanol, 7.5 ml concentrated hydrochloric acid, 33 mg fluorinated phenylmethylsulfonyl (PMSF) and 1 ml aprotinin (Sigma A6012, 0.9% NaCl). And with 9.8 units / ml of trypsin-inhibiting units in 0.9% benzyl alcohol) was mixed with approximately 192 ml of 4 ° C distilled water. According to a preferred embodiment of the invention, recovery of the soluble protein separates the lysed protein from the lysed cells, for example by centrifugation, raising the pH to about 5.0 by, for example, addition of ammonium hydroxide, eg, centrifugation. The precipitate is removed by separation, for example the protein is precipitated from the supernatant by the addition of alcohol, ether or both, for example by allowing the solution to stand at about 4 ° C for about 48 hours and centrifuging. The precipitate is removed from the solution, the organic phase is removed from the precipitate, the precipitate is dried, for example in a fume food, and the dried precipitate is redissolved in a suitable solvent such as 1M acetic acid, eg by dialysis. It consists of a step to remove the low molecular weight solute from the solution. According to a preferred embodiment of the invention, the separate separation and recovery of the polypeptide from the lysate protein is by, for example, a gel hyperchromatography of the protein using a Bio-Gel P-10 resin. According to a preferred embodiment of the invention, the polypeptide assay is individually suitable for human tumor cells such as human lung cancer line (A549) or established mink lung cell line (CCL64) or normal human foreskin fibroblast (HuF). Consists of steps to identify polypeptides that exhibit growth inhibition of human tumor cells or growth inhibition of established minced lung cell line (CCL64) or hyperproliferation of normal human envelope fibroblast by contact with the polypeptide for an appropriate period of time under conditions. .. The method for preparing a substance called tissue-derived growth inhibitor-1 (TGI-1) is to first prepare an acidified ethanol extract and then prepare a high performance liquid chromatographie of the acidified ethanol extract, for example, in reverse phase. According to HPLC chromatographie, (i) about 26-34% acetonitrile linear gradient of acetonitrile containing 0.05% trifluoroacetic acid or (ii) about 17-23% 2 of 2-propanol containing 0.05% trifluoroacetic acid 2 -Consists of recovering TGI-1 as a given activity from the acidified ethanol extract using a propanol linear gradient. A method for preparing a substance called a tissue-derived growth inhibitor (TGI) is to first prepare an acidified ethanol extract and then prepare a high-performance liquid chromatographie of the acidified ethanol extract, for example, a reverse-phase HPLC chromatographie. Therefore, TGI is recovered as a predetermined activity from the acidified ethanol extract using a linear gradient of about 28-34% acetonitrile of acetonitrile containing 0.05% trifluoroacetic acid, or NaCl of about 0.6-0.7 M NaCl. It consists of eluting with a linear gradient and recovering as a single active peak from a cation exchange resin such as CM-trisacrylic. The method for preparing a substance called tissue-derived growth inhibitor-2 (TGI-2) is to first prepare an acidified ethanol extract and then prepare a high performance liquid chromatographie of the acidified ethanol extract, for example, in reverse phase. According to HPLC chromatographie, (i) about 35-39% acetonitrile linear gradient of acetonitrile containing 0.05% trifluoroacetic acid or (ii) about 23-27% 2 of 2-propanol containing 0.05% trifluoroacetic acid 2 -Consists of recovering TGI-2 as a given activity from the acidified ethanol extract using a propanol linear gradient. In the method for preparing heterogeneous aggregates of polypeptides designated CM-I, CM-II, CM-III and CM-IV, an acidified ethanol extract is first prepared, and then an ion exchange chromatograph E, for example, a cation. Peptide heterogeneous aggregates are recovered from the acidified ethanol extract by exchange chromatographie. According to the present invention, a method for detecting the presence of a tumor is disclosed. The method uses TGI-1, TGI, TGI-2 or CM-I, CM-II, CM present in samples collected from subjects such as blood, sheep water, peritoneal fluid, peritoneal fluid, cerebrospinal fluid or urine. It consists of quantitatively measuring the amount of heterogeneous aggregates of polypeptides named -III and CM-IV and comparing this amount with the abundance of the same substance in a normal subject's sample. , A significant difference in this amount, eg, a significant increase, is an indicator of the presence of the tumor. According to the present invention, another method for detecting the presence of a tumor is disclosed. The method presents in a heterogeneous assembly and traits of the polypeptides TGI-1, TGI, TGI-2 or CM-I, CM-II, CM-III and CM-IV present in the subject's sample. Both with the convertible growth factor alpha (TGF-α) were quantitatively measured separately and TGI-1, TGI, TGI-2 or CM-I, CM-II, CM-III and CM-IV in the sample. The ratio of the abundance of the heterogeneous aggregate of the polypeptide designated as to the abundance of TGI-α in the sample was determined, and the same ratio as described above was determined for the sample of the normal subject, and the sample of the subject was determined. Compare the ratio obtained in the above with the ratio obtained in the sample of normal subjects. Significant changes in both values are indicators of tumor presence. According to the present invention, a method for determining a tumor is provided. In this method, a heterogeneous assembly of polypeptides designated TGI-1, TGI, TGI-2 or CM-I, CM-II, CM-III and CM-IV for a sample of a subject with tumor suppression Measure the presence of one or more of them. Specific tumor types such as melanoma or carcinoma can be determined by the presence or absence of specific binding of these components, such as TGI and CM-II or TGI-1 and TGI-2. Finally, another method of tumor typing is disclosed. In this method, there is a heterogeneity of the polypeptides named TGI-1, TGI, TGI-2 or CM-I, CM-II, CM-III and CM-IV in the sample of the subject with the tumor. Quantitatively determine the abundance of aggregates and the presence of each specific or relative amount, such as a significant increase in the amount of TGI, or a significant change in ratio, such as the ratio of TGI-1 to CM-II. The type of tumor is determined from. Detailed description of the experiment Part 1 of the experiment Materials and methods The term "TGI activity" in Part 1 of the experiment means the activity of TGI-1, TGI-2, CM-I, CM-II, CM-III and CM-IV unless otherwise noted. Isolation of tissue-derived tumor growth inhibitors (TGIs) from tissue extracts Davoren et al., Biochemical Biophys. Acta 63: 150 (1962) and Robert et al., Proceedings of National Academic Science (Proc. Natl. Human umbilical cord or placental tissue was extracted using a modified acid / ethanol extraction method described by Acad.Sci.) USA, 77: 3494 (1980). Extractive alcohol is 375 ml of 95 (volume / volume)% ethanol (punctirias, standard strength 190, US Industrial Chemicals, # UN 1170 and 7.5 ml of concentrated hydrochloric acid and 33 mg of fluorinated phenyl. Methylsulfonyl (PMSF) (Sigma P-7627) and 1 ml of aprotinin (with 19.8 units / ml trypsin inhibitory unit in Sigma A6012, 0.9% NaCl and 0.9% benzyl alcohol) with 192 ml of distilled water at 4 ° C. It was a mixture. 400-600 g of frozen human umbilical cord or placenta (Advanced Biotechnologies) (stored at -80 ° C) was thawed at 4 ° C for 6 hours. The tissue was placed in a 4 ° C cooled Cuisinart food processor (model DLC-7-PRO) and suspended in 200 ml of extraction buffer at 4 ° C. The suspended tissue was homogenized with the food processor. The first minute of homogenization turned the suspension into yellowish white (cream white). To this white suspension was added an additional 200 ml of 4 ° C extraction alcohol. The suspension was dark brown. The tissue suspension was homogenized at 4 ° C for a total of 10 minutes. Extraction chloride was added to this homogeneous tissue mixture to give a final volume of 6 ml per 1 g of tissue homogenic solution. The homogeneous structure suspension was transferred to a large 4-beaker equipped with a 3-inch stir bar and stirred with a Lab-line Multimagnestir multi-stage mixer, model # 1278, 1/2 maximum stirring capacity. .. Extract overnight at 4 ° C with agitation, transfer homogenic solution to 1 centrifuge (Sorvall) and in a Sorvall RC-3B centrifuge with a Sorvall H-6000A rotor 4 Centrifuge at 3500 rpm (RCF = 350) at ° C for 30 minutes. The supernatant was transferred to a large 4 beaker and concentrated ammonium hydroxide was gradually added to adjust the pH to 5.0. As the pH rises, the supernatant turns from brown to orange. 2.0M ammonium acetate and pH 5.2 were added to a total volume of 1% to precipitate the solution. Precipitates were removed by centrifugation on Sobal RC-3B at 4 ° C. at 4500 rpm (RCF = 5900) for 4 hours. The supernatant was transferred to a large 6 flask and 4x volume anhydrous ether (-20 ° C) (Baker 9244-3) and 2x volume 95% ethanol (4 ° C) were added. The mixture was allowed to stand at -220 ° C for 48 hours to settle the resulting precipitate. After 48 hours of precipitation, the etherified material was warmed to room temperature in a ventilated hood. Warming the acidified ethanol extract to room temperature promotes precipitation aggregation. The clear organic phase of ether and ethanol was removed with a water aspirator and the precipitate was kept in a ventilation hood for several hours to evaporate the residual organic phase. The "dry" precipitate was dissolved in 1.0 M acetic acid and 1.0 M using a dialysis membrane with an exclusion limit molecular weight of 3500 (Spectropor 3, Spectrum Medical Industries, Los Angeles, Calfornia). Roughly dialyzed against acetic acid (Baker # 9507-5). The dialyzed acidified ethanol extract was lyophilized in a 250 ml Corning conical centrifuge tube (Corning 25350) and stored as an unpurified acidified ethanol extract. As a modification procedure for precipitating TGI from the acidified ethanol extract, instead of adding 4 times the volume of ether and 2 times the volume of ethanol, only 2 times the volume of ethanol may be added at 4 ° C. The advantage of removing the ether from the acidified ethanol extract precipitation steps is that the steps that require a flammable solvent are removed, eliminating the drawbacks associated with the processing and scale-up of large volumes of material. Gel hyperchromatography The lyophilized crude acidified ethanol extract was resuspended in 1.0 M acetic acid (10-30 mg / ml) and distanced for 30 minutes at 3500 rpm at 4 ° C in a Sobal RC-3B centrifuge equipped with a Sobal H-6000A rotor. I clarified it with my heart. The sample is then processed in columns. A 100-150 ml sample was chromatographed on Bio-Gel P10,100-200 Metsyu (Bio-Rad: 150-1040) with 1.0 M acetic acid at 23 ° C or 4 ° C. The column (14 x 100 cm) (Amicon, # 86012) contained 13.8 equilibrated and degassed bio-gel P10 in 1.0 M acetic acid at 23 ° C or 4 ° C. The pore volume was measured by adding 50 ml of 1.0 M acetic acid containing 2 mg / ml blue dextran (sigma # D5751). After calibration, 100 ml of 1.0 M acetic acid containing 100 mg / ml bovine serum albumin (Sigma # A-4503) was added and subsequently washed roughly with 1.0 M acetic acid to "condition" the column. After applying the sample, use a SuperRac (LKB2211) with a C-type collection rack and place each 1 fraction into a 2 plastic tissue culture roller bottle (Falcon, 3207) at a flow rate of 7 ml / min. Collected at. Fractions were monitored with a 280 nm Uvicord S (LKB2138) set to an absorption range of 2.0 AUFS and recorded with a single channel channel trecoder (LKB2210). Partial samples of 1 ml each were taken from each fraction, lyophilized and tested for TGI activity as described below. The rest of each fraction was lyophilized in 2 freeze-dried gears (Virtis # 6503-2050) using a Virtis freezer No. 24. High Performance Liquid Chromatograph E (HPLC) Individual fractions containing TGI activity from the Bio-Gel P10-column were lyophilized and 1-10 ml of 0.05% trifluoroacetic acid (TFA) (Pierce), depending on the amount of protein in each fraction. It was resuspended in # 28901). Water for HPLC was obtained with the Milli-Q water purification system. In all HPLC chromatographies, the starting buffer consists of Milli-Q water containing 0.05% TFA. Prior to injection, the sample was centrifuged in a Beckman desktop centrifuge (Beckman TJ-6) at 3000 rpm for 20 minutes to remove insoluble material. Waters u Bondapak analysis of supernatant C₁₈It was injected into a column (0.39 × 30 cm) (Waters PN27324) or a semi-preparative column (0.78 × 30 cm) (Waters PN84176). Which one to use will be specified in each experiment. A Waters fully automatic gradient controller (Waters 510 type) was used for column elution and monitored with a variable wavelength uv detector (Waters Lambda-max 481 type) set to 206 nm. Acetonitrile (Baker 9017-3) or 2-propanol (Fisher A452) containing 0.05% TFA was used as the elution solvent. Fractions were collected in 13 x 100 mm or 16 x 100 mm silica glass (siliconized) (pierced, Aquasil # 42799) test tubes by SuperRac (LKB2211) with B-type collection rack. .. Partial samples of each collected fraction were taken and TGI activity was tested as described below. Ion Exchange Chromatograph E Both the lyophilized material obtained from the acidified ethanol and ether extracts and the various lyophilized fractions obtained from the Bio-Gel P-10 gel hyperchromatography were separately treated with ion exchange chromatographies. .. CM, SP and DEAE-Tris acrylic (LKB) ion exchange resins were used in these treatments. The chromatographic sample was diluted with 1.0 M acetic acid to a final concentration of about 20 mg / ml. Samples were dialyzed at 4 ° C until both pH and conductivity were equal to the starting (equilibrium) buffer. All ion exchange chromatographies were performed at 4 ° C. a. Ion exchange chromatograph using CM- and SP-Tris acrylic 1.0M resin Suspended as an aqueous suspension in equal volume of 0.1 M ammonium acetate containing NaCl, pH 4.0. The resin was equilibrated for at least 3 hours and degassed at 4 ° C. Fill a 1.6 x 20 cm column (Pharmacia # 19-0362-01) with 20 ml of resin and double the volume of the column with 1.0 M ammonium acetate, pH 4.0, and 0.01 M ammonium acetate, pH 4.0. It was washed sequentially. The column was washed until the effluent matched the equilibrium buffer (eg 0.01 M ammonium acetate, Fisher A637), pH 4.0 exactly. Samples are applied to the resin at a flow rate of 1 ml / min (1 gm / 2 ml resin), washed with an equilibrium buffer until the optical concentration is leveled (eg near 0 optical concentration), concentration 0.01-1.0 M ammoniu acetate, pH 4.0. A 200 ml ascending molar concentration linear gradient (Falmacia gradient mixer GM-1, # 19-0495-01) was applied via the column flow adapter of. In some experiments, the same column was subjected to a second gradient. This second gradient ranged from 1.0 M ammonium acetate, pH 4.0 to 1.0 M ammonium acetate containing 50% acetonitrile, pH 4.0. A 2 ml fraction was collected in a 13 x 100 ml polystyrene tube (Columbia Diagnostics, B2564) with a SuperRac fraction collector (LKB2211) equipped with a type A collection rack. A Uvicord S with a 280 nm filter (LKB2138) and a single channel recorder (LKB2210) was used for all column chromatographies. The fraction was used as a partial sample of 10 μ to 1 ml based on the optical concentration, and the TGI activity was tested. b. DEAE-Chromatographies using Tris acrylic The preparation and treatment of the chromatographie resin were carried out in exactly the same manner as in the case of CM- and SP-Tris acrylic. However, as the equilibrium buffer, 0.1 M ammonium acetate, pH 6.0, was used, and gradient elution in the range of 0.1 M to 1.0 M ammonium acetate, pH 6.0, was used, and the sample was equilibrated with the equilibrium buffer. Single layer of TGI activity 10% bovine fetal serum (Whittaker MABio-products 14-501B) and 2% L-glutamine (Whittaker MABio-Products 17-605-A) ), 1% Penicillin and 1% Streptomycin in 50 μ Dalbetsuco Modified Eagle's Medium (Whittaker M.A. Bio-Products 12-6143) 96-well Tissue Culture Plate (Numc 167008) ), The test cells were subcultured. Human lung cancer cell A549 and normal human human fibrilblast (HuF) are 5 × 10 per well³The seeding density of cells was required. Mink cells (ATCC: CCL64) are 4.5 x 10 per well³The seeding density of cells was required. For TGI activity, transfer a partial sample of the column fraction to be tested to a 12 x 75 mm sterile test tube (Falcon 2058) containing 50 μm 1M acetic acid containing 1 mg / ml bovine serum albumin (BSA: Sigma A-6003). It was lyophilized. Immediately prior to Atssei, lyophilized samples were resuspended to 400 μ for each cell type tested. A 100 μm partial sample of the resuspended sample was added to the test cell containment well. Each sample was tested in 3 sets. 5% CO₂Cells were inked for 72 hours at 37 ° C in a moist atmosphere of / 95% air. At the end of the incubate period, 1 μCi / ml 5-[¹²⁵I] Iodine-2'deoxyuridine (¹²⁵Each well was pulsed for 24 hours in 100 μm complete medium containing IUdR) (New England Nuclear; NEX-072). Wash monolayer Batuhua A (Dalbetsuco Phosphate Buffered Saline, 100 mM MgCl₂, 1 mg / ml BSA content, pH 6.8), fixed in methanol (Fitzerer A452) for 10 minutes and air dried for 15 minutes. Incorporated into cells¹²⁵IUdR was solubilized with 200 μl 1.0N NaOH and the plate was inked at 60 ° C. for 20 minutes. Solubilization¹²⁵IUdR was collected at the Titertek Supernatant Collection System (Scatron Inc., 7072). The amount of cell proliferation was incorporated into the DNA of the cell during the logarithmic growth phase.¹²⁵Estimated according to the degree of IUdR. Prior to Atssei, each well collected was observed with a Zeiss inverted microscope and the amount of cell proliferation was measured with the naked eye. Inhibition or enhancement of proliferation was incorporated into test cells (eg, human tumor cells) containing the test sample.¹²⁵Incorporated into IUdR and untreated control cells¹²⁵It is shown as a ratio with IUdR. Increased or inhibited treatment cells when observed under a microscope¹²⁵It was consistent with the increase / decrease in the amount of IUdR uptake. Trait TGI activity a. Heat treatment 1 ml partial samples of fractions 2, 4 and 6 obtained by gel hyperchromatography of Bio-Gel P-10 were lyophilized in a 12 x 75 mm polystyrene tube (Falcon 2034) and reconstituted in 1 ml of 1.0 M acetic acid. Suspended. The sample was heated in a boiling water bath for 3 minutes, lyophilized and tested for TGI activity in the same manner as described above. b. Treatment with dithiothreitol (DTT) A 3 ml aliquot from fractions 2, 4 and 6 obtained as a result of gel hyperchromatography of Bio-Gel P-10 in a 17 x 125 mm polystyrene screen (Columbia) Freeze-dried in Diagnostics) # 2570). To the treated sample, 0.1 M ammonium hydrogen carbonate 200 μ, pH 7.4 containing 0.1 MDDT (Cal-biochem # 233-153) was added at a final concentration. Control samples contained 200 μm of 0.1 M ammonium bicarbonate (Fisher, A643, pH 7.4. These samples were inked at 23 ° C for 1 hour, diluted to 1 ml with 1.0 M acetic acid and 3,500. Dalton's molecular weight cut-off 18 mm dialysis membrane (Spectropor 3, Spectrum Medical Industries) was used to dialyze 0.1M acetic acid 4 with 4 changes. These samples were 12 x 75 mm. It was lyophilized in a polystyrene tube (Falcon # 2304) and TGI activity was analyzed as described above. Slab gel electrophoresis of SDS-polyacrylamide The aliquots from the samples obtained by each chromatographic treatment were lyophilized for electrophoresis. These samples were diluted with 0.1 M Tris-HCl (Sigma; T-1503), pH 6.8, 15% glycerol (Kodak; 114-9939), and 2% sodium dodecyl sulfate (SDS). Dilute the sample to 80μ of sample containing (Bio-Rad; 116-0302) and 5% 2-mercapto-ethanol (Bio-Rad; 161-0710), essentially by the method described above. Electrophoretic treatment with a 5-20% acrylamide monomer gradient (Laemmli, UK (1970) Nachy magazine 227,680-685). Boil these samples for 2 minutes, then apply a constant current of 30 mA / gel (Hoeffer power supply; PS1200DC) for 4 hours at 9 ° C in a bio-rad model 155 vertical electrophoresis cell (bio-rad 165-1420). Placed on a 1.5 mm wide slab gel. The temperature was maintained constant by a water bath circulator (Haake, A81). The gel was placed in 5.7% acetate and 47% methanol. 0400) Colored overnight with 0.5% and then decolorized with the same solution without colorants as above. Specific gels with low protein concentrations were silvered by Merril (Merril, CR, Gold). Recolored by Man, Day (Goldman, D.), Sedman, S. and Ebert, MH (1981) 211; 1437-1438) (Bio-Rad, Silver staining kit; # 161-0443). Result Comparison of TGI activity resulting from gel hyperchromatography on bio-gel P10 at room temperature and 4 ° C The growth inhibitory activity derived from the acidified ethanol extract of the human umbilical cord was apparently eluted with a molecular weight of 5,000 to 16,000 daltons by gel hyperchromatography using a bio-gel P.10 resin. Occasionally, another active peak was also observed with a molecular weight of 3,000 to 5,000 daltons. The calculation of molecular weight is based on the elution profile of the molecular weight standard (ie carbonic anhydrase-29,000; RN Aasee 14,400; insulin-6,000) chromatographed with 1 resin in a 4 x 100 cm column. The elution profile obtained by the column and the profile obtained by the 14 × 100 cm large column could be superposed on each other. The acidified ethanol extract from human placenta chromatographed in the same manner showed an elution profile very similar to the umbilical cord extract. Fractions 1-3 from the umbilical cord acidified ethanol extract have a very dark brown color, but this color gradually disappears as the fraction progresses. Fortunately, although tumor cell growth inhibitory activity (TGI) is eluted in fractions 1, 2 and 3 with the highest protein concentrations, most of this activity is from Figures 1 and 2. As is clear, it extends beyond the observed protein peak. Extracts from human placental material had a greater TGI overlap with major protein peaks than was observed for material from the human umbilical cord (data not shown). Multiple equal aliquots from gel hyperchromatographic electrophoresis by SDS-PAGE on a 5-20% polyacrylamide gradient also found that fraction 4 detected significantly less protein than fractions 1-3. Indicated. In fractions 5 and 6, 5600 and 14,000 major protein bands were observed, and in fraction 7, although the inhibitory activity extended to fraction 10 as shown in Fig. 2, only a very small amount of protein remained. A significant advantage of the large activity eluting into the region of less protein is that it facilitates a higher degree of purification of TGI. Comparing the bio-gel P10 chromatograms at room temperature shown in Figures 1 and 2, respectively, with those at 4 ° C, the inhibitory activity is clearly better conserved at 4 ° C. At 23 ° C, no activity was observed above fraction 6 (Fig. 1), but at 4 ° C the activity was further extended by 4 fractions to reach fraction 10. Most importantly, the net amount of activity recovered is at least twice as large when the extract is chromatographed at 4 ° C. This is because TGI activity of 80% or more can be obtained in 7 fractions at 4 ° C (Fig. 2), whereas it can be obtained in only 3 fractions at 23 ° C. This is clearly due to the fact that the same amount of activity elutes in the three fractions (23 ° C) spreads over the seven fractions (4 ° C), but is clearly a practical yield of TGI activity. It was due to the increase. Fraction 5 1 ml aliquots obtained separately from the two columns and 1/5 to 1/125 dilutions of these fractions were tested for human lung adenocarcinoma (A549) and mink lung cells (CCL64) ( Table I). The TGI activity of the undiluted fraction was twice as high in the fraction 5 obtained by chromatographic at 4 ° C. In addition, the 25-fold dilution of Fraction 5 by chromatography at 4 ° C continued to show maximum TGI activity against human tumor cell lines. Fractionation of equivalent dilution by chromatographi at 23 ° C showed no detectable activity. Similar observations were made for the mink cell line. This data is not based on the activity seen in Figures 1 and 2, but is obtained from two separate columns showing comparable TGI activity in each fifth fraction. Comparison of the effect of TGI on normal human fibroblasts (HuFs) and the effect of TGI on human transformed lung cancer cells (A549) Aliquots of fractions obtained from human umbilical cord acidified ethanol extracts chromatographed with Bio-Gel P-10 resin at human umbilical cord acidified ethanol were subjected to human normal cells and human traits as described in the Materials and Methods section. A TGI activity test was performed on transformed cells. As is clear from Fig. 3, the TGI activity on human A549 cells (white triangles) ranges from fraction 3 to fraction 12, but these fractions increase by 85% in the growth-stimulating effect of normal human human fibroblast. I let you. As described above, the inhibitory activity is specific to human tumor cells. This observed inhibitory activity is not due to cytotoxicity, as shown by photomicroscopy and indirectly by stimulatory effects on normal human fibroblasts. TGI was previously tested on normal epithelial-derived cells and similar effects were observed.<img file="JPH0788397B2_D0001.tif" /><img file="JPH0788397B2_D0002.tif" />TGI activity analysis was performed using 1 ml aliquots (Figs. 1 and 2) with gel excess of Fraction 5 containing 120 μg. High Performance Liquid Chromatograph (HPLC) Partially purified by geling on a bio-gel P10 column, followed by reverse phase HPLC (u Bondapak C)₁₈TGI from acidic ethanol extract of human umbilical cord further purified using (resin) suppressed the growth of both A549 human cancer species and established mink lung cell line CCL64, but suppressed the growth of normal human fibroblast. I didn't know. Figure 4 shows the TGI activity obtained by HPLC using a linear acetonitrile gradient of lyophilized fraction 4 (19.8 mg / 3 ml 0.05% trifluoroacetic acid) obtained from the Bio-Gel P-10 Chromatograph Istep. Shows the elution profile. A549 It was observed that there were two distinct peaks in growth inhibitory activity on both human cancer types and mink cells. Fractions eluted with 28-34% acetonitrile (fractions 13-22) and fractions eluted with 35-39% acetonitrile (fractions 25-31) are pooled separately and a 2-propanol linear gradient. Using C₁₈u Chromatographed again on a bonder pack column. The first peak of TGI activity is called TGI-1, and the second peak is called TGI-2. Figure 5 shows the elution profile and TGI activity of TGI-1 (Figure 4). The concentration of injectable material was 1.5 mg per 1.5 ml of 0.05% trifluoroacetic acid (TFA). TGI-1 activity elutes between 17-23% using a 2-propanol linear gradient. Similarly, Fig. 6 shows that TGI-2 (0.8 mg / 1.8 ml 0.05% TFA) (Fig. 5) is between 23 and 27% (fractions 17 to 23) using a 2-propanol linear gradient. It shows that it is rechromatographically processed. The TGI activity in Figures 4 and 5 is consistently 20% higher for mink cells than for A549 human cancer cells. Another total fraction from the Bio-Gel P-10 chromatograph set, fraction 5 (see Figure 3), was chromatographed by HPLC using an acetonitrile linear gradient (Figure 7). Fraction 5, which is a fraction after the material used in Fig. 4 (fraction 4), mainly contained inhibitory activity that elutes as TGI-1. In Fig. 7, TGI-1 was eluted with 29-34% acetonitrile. Growth inhibitory activity was 35% higher for mink cells than for A549 human cancer cells. Here most of the TGI activity was separated from the protein peak. The active fractions (14-25) were pooled, lyophilized and chromatographed by reverse phase HPLC using a 2-propanol linear gradient (Fig. 8). Evidence of differential inhibitory activity was observed. TGI activity is 23% 2-propanol (fractions 22-25) elution with mink cell-specific growth inhibitory activity and 26% 2-propanol (fractions 25-25) elution with A549 human cancer. It was divided into seed cell-specific growth inhibitory activity. Therefore, the fractions that are later eluted by gel hyperchromatography and have less contaminating proteins [less absorbance at 280 nm (Figure 2) and less protein by gel electrophoresis] are two different. Has cell-specific inhibitory activity. The acidic ethanol extract of human placenta uses a linear acetonitrile gradient containing 0.05% TFA after gel hyperchromatography isp C.₁₈It also contained TGI activity that eluted with 26-34% acetonitrile in the column. Ion exchange chromatograph 1 g of lyophilized acidified ethanol extract of human umbilical cord was directly ion exchange chromatographed with CM-TRISACRYL in 0.01 M ammonium acetate, pH 4.0. A linear gradient of ammonium acetate from 0.01 to 1.0 M, pH 4.0 was applied. Figure 9 shows at least four distinct TGI activities, CM-I, CM-II, CM-III and CM-IV. At that time, CM-I suppressed only A549 human cancer cells with a suppression rate of 60% (Table 2). CM Peaks II and III show similar growth inhibitory activity against A549 human carcinoma (80% and 63%, respectively) and against mink cells (61% and 76%, respectively). The final activity peak (CM-IV) was activity-specific for mink (ie, mink cell inhibition (95%) was higher than A549 human cancer cell inhibition (69%)). When a negatively charged resin was used, CM-I was not retained, and CM-II was slightly delayed. This is because both CM-I and CM-II were eluted with 0.01M ammonium acetate and pH 4.0 before the gradient was initiated. All proteins with inhibitory activity are acidic proteins because they can dissolve at pH 4.0 and bind to negatively charged resins, but peaks CM-III and IV are eluted with CM-Tris acrylic resin (0.5M or more ammonium acetate). ) Is more strongly bound, so it is probably a little more basic. This is demonstrated by the fact that there was no TGI activity retained by the positively charged resin (ie DEAE-Tris acrylic) (data not shown). The more acidic inhibitors appear to have greater specificity for each activity on A549 human carcinoma cells. These four TGI activity peaks (CM-I, CM-II, CM-III and CM-IV) were repeatedly observed (six separate chromatographic treatments with CM-Tris acrylic). CM-III to prevent the TGI activity observed in CM-III and CM-IV from producing substances that can be eluted earlier from the column, and to support the notion that each activity peak is a separate entity. And the material from CM-IV was pooled, lyophilized and chromatographed again using CM-Tris acrylic under the same conditions as the column from which this material was derived. CM-III and CM-IV were eluted at exactly the same positions as the original column fractions that induced them (greater than 0.5M ammonium acetate) (Fig. 10). The higher TGI inhibitory activity on mink cells was retained, and the difference between the inhibitory activities on the two cell lines was maintained exactly the same at 25-30%, centered on the activity peak. Physical and biological characteristics of tissue-derived tumor cell growth inhibitory activity (TGI) Fractions 2, 4 and 6 obtained as a result of gel hyperchromatography with Bio-Gel P10 were either heat treated (Table 3) or reduced by dithiothreitol (DTT) treatment (Table 4). All of the fractions tested retained TGI activity after heat treatment or acid treatment (see Table 5). Fractions 2, 4 and 6 were found to suppress the growth of human cancer cells and stimulate the growth of normal human cells. The TGI activity of fractions 2 and 6 decreased when treated with DTT, but the TGI activity of fraction 4 increased slightly (Table 4). This data also demonstrates the existence of a separate TGI.<img file="JPH0788397B2_D0003.tif" />The protein concentration of the fraction subjected to the TGI activity test was 15 to 300 μg.<img file="JPH0788397B2_D0004.tif" />The protein concentration of the fraction subjected to the TGI activity test was 15 to 300 μg.<img file="JPH0788397B2_D0005.tif" />The protein concentration of the fraction subjected to the TGI test was 5 to 300 μg.<img file="JPH0788397B2_D0006.tif" />Part 2 of the experiment Materials and methods Separation of tissue-derived tumor growth inhibitors (TGIs) from tissue extracts from which blood, veins and arteries have been removed Veins and arteries were removed from human umbilical cord tissue, the remaining tissue was thoroughly washed to remove blood, and then acid / ethanol extraction was performed as described in the first part of the experiment. The public address system (PBS-PA) used for washing and homogenizing this tissue was NaCl 16 g and Na.₂HPO₄ H₂O2.5g and NaH₂PO₄ 7H₂Sigma A6012 with 0.4 g of O, 116 mg of phenylmethylsulfonyl (PMSF) (Sigma (Sigma) P7627), and 19.8 units / ml of trypsin inhibitor in Aprotinin (0.9% NaCl and 0.9% benzyl alcohol). ) And water 2 was adjusted to pH 7.4 with HCl and NaOH. Extracted buffer is 95% (v / v) ethanol (punk chilimus, 190 proof, US Industrial Chemicals, # UN 1170) 375 ml, concentrated HCl 7.5 ml, and hutuated phenylmethylsulfonyl (PMSF) (sigma). P-7627) 33 mg and 1 ml of aprotinin (Sigma A6012) were mixed with 192 ml of distilled water at 4 ° C. 800-1,000 g of frozen human umbilical cord (Advanced Biotechnology) Biotechnologies); stored at -80 ° C) was thawed by immersing in PBS-PA at 4 ° C for 2 hours. Individual umbilical cords were removed and washed with PBS-PA. Veins and arteries were removed from the umbilical cord by incision at 4 ° C. The dissected umbilical cord was washed with fresh PBS-PA to remove residual blood and vascular debris. This tissue was placed in a 4 ° C cooled Cuisinart food processor (model DLC-7-PRO) and suspended in 200 ml of 4 ° CPBS-PA. This suspended tissue was homogenized with the food processor. After the first 1 minute of homogenization, an additional 200 ml of 4 ° CPBS-PA was added. The tissue suspension was homogenized at 4 ° C for a total of 10 minutes. The resulting homogenate was transferred to a 200 ml centrifuge bottle (Sorvall) and centrifuged in a Sobal RC5B centrifuge equipped with a Sobal GSA rotor at 4 ° C for 5 minutes at 9000 rpm (RCF = 13,000). The supernatant fluid was removed and discarded and the pellet was resuspended in fresh PBS-PA to the original homogenate amount. The pellet was washed by repeating centrifugation and resuspension as described above until the supernatant fluid became clear and the red color from the contaminated blood or blood products disappeared. The pellet obtained after washing was white. The washed pellet was resuspended in extraction buffer at a final volume of 6 ml per gram of original cut tissue. The resulting homogenate was transferred to 4 large beakers with a 3-inch stir bar and stirred with a LAB-line Multimagnestir multimixer, 1/2 of the maximum stir capacity of model # 1278. After overnight extraction with stirring at 4 ° C, the homogenate is transferred to a 1-centrifuge bottle (Sobal) and placed in a Sobal RC-3B centrifuge equipped with a Sobal H-6000A rotor at 4 ° C for 30 minutes at 3500 rpm (RCF). = 3570) was centrifuged. The supernatant was transferred to 4 large beakers, and the pH was adjusted to 5.0 while slowly adding concentrated ammonium hydroxide. The supernatant remained pale yellowish and transparent as the pH was increased. Ammonium acetate 2.0M solution, pH 5.2 was added in an amount of 1% of the total amount. The precipitate formed at this step was completely removed by centrifugation with Sobal RC-3B at 4 ° C for 4 hours at 4500 rpm (RCF = 5900). The supernatant was transferred to a 6-size flask, to which 4 times the volume of anhydrous ether (-20 ° C) (Baker # 9244-3) and 2 times the volume of 95% ethanol (4 ° C) were added. The mixture was left at rest at -20 ° C for 48 hours to allow the formed sediment to settle. After precipitation for 48 hours, the material was brought to room temperature in a fume hood. Warming the acidified ethanol extract to room temperature promotes aggregation of the precipitate. The clear organic phases of ether and ethanol were removed with a water aspirator and the precipitate was left in the ventilation hood for several hours to evaporate the residual organic phase. A gentle stream of dry nitrogen gas over the extract promoted evaporation of the residual organic solvent present with the precipitate. Dissolve the "dried" precipitate in 1.0 M acetic acid and use a molecular weight cut-off 3500 dialysis membrane (Spectropo-3, Spectrum Medical Industries, Los Angeles, California) to 1.0 M acetic acid (Baker # 9507-5). On the other hand, it was sufficiently dialyzed. The acidified extract after dialysis is lyophilized in Corning's 250 ml conical centrifuge tube (Corning 25350) and stored as a crudely acidified ethanol extract, or 20 mM NH.₄O₂C₂H₃, pH 4.5 was carefully dialyzed. Comparison of the TGI activity of the first acid / ethanol extract from the treated tissue as described in Part 1 of the experiment with the TGI activity in the case of the treated tissue as described above. Table 6 shows the increase in specific activity and total recovery activity observed when the tissue was treated as described above. This table shows the proteins and proteins related to the frozen umbilical cord treated according to the procedure described in Part 1 (hereinafter referred to as the "first procedure") and the frozen umbilical cord treated as described above (hereinafter referred to as the "improved procedure"). This is to compare the yield of TGI activity. There are some significant differences between these two procedures for later TGI purification. For example, based on the wet weight of tissue, acidified ethanol extraction by the first procedure recovers 0.33% as protein (3.3 g from 1000 g of tissue), whereas according to the improved procedure 0.015% as protein (340 g). Only 0.05g) is extracted from the tissue. Active yield when using the improved procedure (3.3 x 10)⁶Units) are obtained from 66% less tissue (340g vs. 1000g) than in the first step and the yield (2x10) obtained in the first step.⁶Since it is 50% larger than the unit), the overall extraction efficiency has increased. The first procedure yielded 2000 units of TGI activity per gram of umbilical cord (wet weight). The improved method yielded 9700 units of TGI activity per gram (wet weight) of umbilical cord. The overall extraction efficiency increased 5-fold according to the improved procedure. Also, because less protein was extracted with acidified ethanol, less ether and ethanol were required to precipitate the extracted protein. In addition, the improved procedure will determine the amount of protein extracted.<img file="JPH0788397B2_D0007.tif" />And because of the smaller number of protein types, less chromatographic material, shorter treatment times, and fewer steps can be obtained in the purification process for later use. Fractionation of TGI extracted by the modified procedure with the cation exchange resin CM-trisacryl was eluted as a single peak from most of the applied protein when the binding material was eluted with a linear gradient of 0-1.0 M NaCl. FIG. 11 shows the fact that when TGI was applied to the CM-trisacryl column, no inhibitory activity was detected in the materials not bound to this resin (ie, fractions 1 to 24). When NaCl was added linearly while increasing the amount (-), most of the proteins bound to the resin (fractions 25 to 38) were removed, followed by a significant amount of inhibitory activity ( ... , fraction 39). ~ 49) has been removed. The most effective NaCl concentration for removing bound TGI was about 0.6 M (fraction 44). As can be inferred from the comparison between Fig. 11 and Fig. 10, the inhibitory activity eluted in the experiment in Fig. 11 is the salt concentration for elution (NaCl in Fig. 11 and NH in Fig. 10).₄O₂C₂H₃) Are similar to each other (0.6M in Fig. 11 and 0.6 to 0.7M in Fig. 10), so the elution of CM-III and CM-IV from CM-Tris acrylic as shown in Fig. 9 Seems to be very close to. This data also suggests that treatment of the tissue by an improved procedure preferentially isolates the single type TGI, thus improving the subsequent characteristics of the factor. Another feature of TGI extracted from the tissue by the improved procedure is that it does not bind to the anion exchange resin. Figure 12 shows the results of adjusting the pH to 8.0 as described in the description section of this figure and applying the extract to the cation exchange resin DEAE-Tris acrylic (the same amount as in Figure 11). It was shown that most of the inhibitory activity corresponded to unbound substances (fractions 1-30), while most of the applied proteins (defined by absorbance at 280 nm) (-) were bound to the column resin. There is. These results mean that contaminating proteins can be removed from the TGI under the conditions of FIG. 12 and are therefore a useful method for TGI purification. These results further indicate that TGI is a cation because it does not bind to the anion exchange resin at pH 8.0. In addition, the TGI extracted by the improved procedure has ionic properties, and the polypeptides extracted by the ion exchange resin in the first procedure (GTI-1, TGI-2, CM-I, CM-II, CM-III and It is also found from the results in Fig. 12 that it is similar to CM-IV). This is because none of the polypeptides bind to the anion exchange resin. Large volumes of samples can be reproducibly separated by CM-trisacryl, thus providing more TGI for subsequent purification processes. In Figure 13, CM-Tris with 9.9 mg of tissue extract under the same chromatographic conditions as in Figure 12, using a smaller sample size (2.65 mg protein) and a smaller CM-trisacryl column (5 ml). It was applied to an acrylic column (15 ml). Elution of TGI activity from most protein substances by the NaCl linear gradient was essentially the same in both of these two experiments. FIG. 14 shows the separation of pooled samples (Fig. 13, fractions 59-78) from the CM-trisacryl column by HPLC using the u Bondapack C18 column. No significant inhibitory activity was observed even when the acetonitrile concentration was linearly increased from 0 to 25% after sample application. However, the TGI activity for both A549 (human lung cancer) and CCL64 (mink lung, 0-0) elutes in a single peak with 28-34% acetonitrile (fractions 21-31), while absorbing at 206 nm. Most of the material was eluted with lower acetonitrile concentrations (fractions 11-19) and higher acetonitrile concentrations (fractions 37-50). The fact that the active profile, the absorbance at 206 nm, and the acetonitrile concentration that effectively elutes the TGI activity are similar between Figures 7 and 14, is that the TGI separated by the improved procedure is experimental. It strongly demonstrates that it is similar to or the same as what was named TGI-1 in Part 1. TGI (referred to as TGI-1, CM-III and CM-IV in the first step) by gel hyperchromatography (Cefadex G-50, data not shown) using appropriate protein criteria of known molecular weight. The apparent molecular weight was measured. In the absence of certain interfering proteins (eg hemoglobin), the TGI apparent molecular weight was found to be between 20 kDa and 30 kDa under non-denaturing conditions. The improved procedure detailed here represents a powerful and simple method for removing the Inactive or interfering compounds from the TGI-containing extracts prepared as described in the first procedure. This improvement procedure also improves the efficiency of the various chromatographic operating steps used to separate TGIs by reducing the amount of chromatographic material required and thus reducing the processing time of the TGI. In addition, as described above, the TGI extraction method from the umbilical cord as described here allows the TGI and other proteins to be chromatographed with higher reproducibility than the procedure described above. The characteristics of the TGIs separated according to the improved procedure were investigated for the chromatographic characteristics of both the reversed-phase high performance liquid chromatograph and the CM-Tris acrylic ion exchange chromatograph. TGI has similar or similar properties to TGI-1 by rp-HPLC (compare Figures 7 and 14) and therefore has similar or equivalent hydrophobicity and is a cation exchange resin. It was found to show similar or same properties as CM-III and CM-IV (compare Figures 9 and 11) and therefore have similar or equivalent ionic properties. Therefore, TGI separated by the improved procedure, TGI-1, and CM-III and CM-IV are similar or the same compounds having similar or equivalent ionic properties and hydrophobicity, and thus have similar or the same composition. Is concluded to have. Therefore, the improved procedures described herein provide a more effective way to obtain the purer form of TGI required for more advanced analysis and feature testing.

[Simple explanation of drawings]

1 to 14 are diagrams showing the elution pattern of the active ingredient from the extract of the present invention under various conditions.