Method for identifying and enriching cell specific target structures

The present invention relates to a method for the identification and enrichment of cell-specific target structures, in particular for the identification of cell-specific protein combination patterns on the surface of cells and for the enrichment of these cells.

The identification of cell-specific target structures is of central importance in the Elucidation of cell-cell interactions, the countless effects within an organism may give rise to. In particular, knowledge disease-specific target structures is a crucial for the development of effective and at the same time less adverse effects Drug.

It is known that immune cells (lymphocytes), specific combinations of proteins, also protein combination patterns or shortly called PKM expressing that in a bond Endothelial cells of the blood vessels of the brain and muscle tissue are responsible. Other Combinations of proteins not lead other hand capable of binding to these endothelial cells. Surprisingly, these specific combinations are inter-individually constant and always have the same binding functions. It appears therefore in the specific Proteinkombinationsmustem a interindividual constant lymphocyte binding code to act the cell surface for organ-specific endothelial cell surface, of a represents cell-specific target structure. Cell-specific target structures may thus quite exhibit specific protein combination patterns.

Invasive tumor cells have specific protein combination patterns on their Cell surfaces that touch to a targeted, ie organ-selective invasive behavior. Such Protein combination patterns therefore represent targets for potential drugs.

but is sine qua non for the development of such highly selective drugs Understanding the molecular composition of these target structures.

A method for identifying target structures are known from the prior art known to the on the analysis of gene expression profiles of diseased tissues or cells as compared based on gene expression profiles of healthy tissues or cells, both Protein expression profiles and expression profiles of messenger ribonucleic acid (mRNA) Light on the appearance of new proteins, dysregulated or abnormally modified proteins to give in diseased tissues or cells (eg, in: F. Lottspeich / H.Zorbas; bioanalysis; Oxford University Press; Heidelberg, 1998).

However, these methods all start from cell homogenates, which usually thousands or Millions of cells are based, because only with the help of this cell amounts expression profiles of the above type can be created. In the cell homogenates the cells lie in more open form, so that the proteins or mRNA molecules using biochemical can be extracted and separated method.

These known methods are not suitable, protein combination patterns to identify, since the individual protein components of such a protein combination pattern by the generation of cell homogenates and by the subsequent extraction processes are completely separated and the essential information regarding their cell- and gewebetopologischen location is lost. can by destroying the cell compartments Furthermore, no information regarding the combinations of proteins within this Cell compartments and their relative topological relation obtained another.

Another disadvantage of the known method is that the preparation steps of Tissue or cells of their removal or extraction to the step of isolating or separation of proteins of a large number of variable external influences may be subject, which are difficult to control and unify.

Another disadvantage of the known method is, moreover, that no analysis on the is the single cell level feasible, so that differences between the individual cells with respect to their Protein combination patterns are undetectable. In addition, proteins can only small Amount present, will not be detected by the known methods. this concerns in particular proteins or specific protein combinations, for example, only in few, however, pathogenic, disease-specific cells vorkommmen.

The object of the invention is therefore to provide a method of the abovementioned type, by can be identified and enriched cell-specific protein combination patterns and by which the mentioned disadvantages of the prior art are overcome.

This object is achieved by an inventive method for identifying and Enhancement of cell-specific target structures, in particular for the identification cell-specific protein combination patterns on the surface of cells and for Enrichment of these cells with the features of claim 1.

Advantageous embodiments of the inventive method are in the dependent claims described.

An inventive method for identifying and enriching cell-specific Targets, in particular for the identification of cell-specific protein combination patterns on the surface of cells, and for enriching these cells, comprising the steps of: (A) applying a heterogeneous cell mixture onto one or more surfaces with predefined structures, cells with appropriate target structures on the surface or the surfaces to be bonded; (B) removing the non-binding cells of the Cell mixture from the surface or surfaces; (C) identifying the cell-specific Target structures, which are responsible for binding of the cells to the surface or surfaces; (D) selection and enrichment of cells with identical cell-specific target structures on the surface or surfaces; and (e) the biochemical characterization according to method step (D) selected targets. Thereby, it is possible that those cell-specific Target structures, especially protein combinations the surface of cells identified may be responsible for binding to predefined structures. moreover these cell-specific target structures such as single cell surfaces enriched are to then deliver biochemical analysis method. Thus, proteins that only present in small quantities, are enriched and analyzed. This concerns in particular Proteins or specific protein combinations, for example, only in a few, however, pathogenic, disease-specific cells vorkommmen. In this way, ie, cells one and the same type of function in relation to highly selective binding for further Analyzes of them expressed proteins provided. Advantageously, the biochemical characterization of selected and enriched targets under Method step e) by means of a molecule or molecule complex separation method, such as a Protein separation process, in particular a 2-D gel electrophoresis.

An embodiment for carrying out the process steps (a) to (b) of the invention includes the following part process: 1) input and applying the liquid, heterogeneous Cell mixture in a channel of an analysis apparatus and to a microscope slide with a fixed, a predefined structure contained surface; 2) removal of the non-binding Cells via a channel opening of the passage and collecting said material in a vessel; 3) Cooling of the slide by a slide thermostat, and 4) of the fixation on the Slides bound cells.

In an advantageous embodiment of the inventive method is the heterogeneous Cell mixture from human or animal tissue or human or animal Body fluids isolated or cultured cells are attached and the surface is a human or animal tissue section and / or endothelial cells and / or protein chips and / or a pre-bred human or animal tissue piece. This is a rapid and accurate identification of cell-specific target structures for the development of Medicines guaranteed.

In a further advantageous embodiment of the inventive method is the Identifying the cell-specific target structures with a method carried out, the comprising the steps of: (I) automated application of a reagent solution Y1, the comprises at least one marker molecule to the cell-specific target structure; (II) exposing the Reagent solution Y1 and automated detection of at least one marking pattern of having the reagent solution Y1 labeled target structure; (III) removing the reagent solution Y1 before or after detection of the marking pattern, and repeating steps (I) and (II) with further reagent solutions Yn (n = 2, 3 ... N), each of the at least one marker molecule and / or at least one other marker molecule; and (TV) Summarize respectively in step ii) the detected marker pattern to a complex molecular Combination patterns of cell-specific target structure. With this identification method rapid and accurate identification of cell-specific target structures such as protein combination patterns guarantees on cell surfaces.

In a further advantageous embodiment of the inventive method after the Step (d) the following method step is carried out: (dl) performing Inhibition experiments with respect to one or more components in process step (d) selected cell-specific target structures for detecting a binding hierarchy of Components, wherein the components of one or more proteins of a cell-specific Protein combination pattern are. This can be determined which of these proteins selective binding protein in the hierarchy are particularly relevant and which should not. relevant Proteins are targets for the development of highly selective drugs.

In a further advantageous embodiment of the inventive method instead of process step (e) the following steps carried out: (f) Automated Applying a reagent solution Y1 having at least one marker molecule, to selected and enriched cell-specific target structure; (G) exposing the reagent Y1 and automated detection of at least one marking pattern of the Reagent solution Y1 labeled target structure; (H) removing the reagent solution Y1 before or after the detection of the marking pattern, and repeating steps (f) and (g) with further Reagent solutions Yn (n = 2, 3 ... N), the or each said at least one marker molecule and / comprise at least one other marker molecule; and (i) combining the respective in Step (g) detected marker pattern to a complex molecular combination pattern of selected and enriched cell-specific target structure. According to enriched and selected cells of the same type permit now that the means Process steps (f) to (i) the cell-specific target structures still much more accurate can be investigated.

In summary, the inventive method allows, among other things, that for the Drug development relevant valid targets can be found efficiently, by biological Selektivitätsmechanimen for the identification of target structures underlying be placed.