EP1573053A2

EP1573053A2 - Method for determining the homeostasis of hairy skin

Info

Publication number: EP1573053A2
Application number: EP03767789A
Authority: EP
Inventors: Dirk Petersohn; Kordula Schlotmann; Thomas Gassenmeier; Olaf HOLTKÖTTER; Marcus Conradt; Kay Hofmann
Original assignee: Henkel AG and Co KGaA
Current assignee: Henkel AG and Co KGaA
Priority date: 2002-12-20
Filing date: 2003-12-11
Publication date: 2005-09-14
Also published as: US20060088852A1; WO2004059002A2; DE10260931A1; WO2004059002A3; AU2003292230A1; DE10260931B4

Abstract

The invention relates to a method for determining the homeostasis of hairy skin in vitro, to test kits and biochips for determining markers of hairy skin, in addition to the use of proteins, mRNA molecules or fragments of proteins or mRNA molecules as markers of hairy skin. The invention also relates to a test method for detecting the effectiveness of cosmetic or pharmaceutical active substances for treating hairy skin in addition to a screening method for identifying cosmetic or pharmaceutical active substances for treating hairy skin and to a method for producing a cosmetic or pharmaceutical preparation for treating hairy skin.

Description

Procedure for determining the homeostasis of hairy skin

The present invention relates to a method for determining the homeostasis of hairy skin in vitro, test kits and biochips for determining markers of hairy skin and the use of proteins, mRNA molecules or fragments of proteins or mRNA molecules as markers of hairy skin; furthermore a test method for the detection of the effectiveness of cosmetic or pharmaceutical active substances for the treatment of hairy skin as well as a screening method for the identification of cosmetic or pharmaceutical active substances for the treatment of hairy skin and a method for the production of a cosmetic or pharmaceutical preparation for the treatment of hairy skin.

Every living cell is able to react to signals from its environment. The reactions of the cells are realized by an orderly regulation of the gene expression, so that the metabolism of cells is not static but very dynamic. According to recent estimates, the human genome contains between 30,000 and 140,000 genes. Of this immense amount of information, however, each cell uses only a small, specific part for the synthesis of proteins, which is reflected in the gene expression pattern. Exogenous signals are received by cells and lead to changes in the gene expression pattern, partly via complex signal transduction cascades. In this way, every cell responds to signals from its environment by adapting its metabolism.

In addition to this relatively short-term change in gene expression, every living cell is subject to the aging phenomenon, a process that is associated with the slow change in gene expression.

Human skin is the largest organ in the human body. It is a very complex organ, which consists of a variety of different Cell types exist and form the body's interface with the environment. Most skin cells are found in the epidermis and dermis.

Skin appendages such as Hair follicles, sebaceous glands, sweat glands etc. are formed by a smaller proportion of specialized cells. For example, only less than 5% of the skin cells are involved in the hair follicle structure. This fact makes it clear that the cells of certain skin appendages are difficult to carry out biological analyzes, e.g. Gene expression analyzes can be subjected. In order to understand the reactions of the skin and especially its appendages to exogenous stimuli, the analysis of gene expression is of crucial importance.

Isolation of the skin appendages is technically difficult to implement and very time-consuming. Furthermore, for a realistic representation of biochemical processes in the skin or its appendages, it is essential to examine the cells in their natural, cellular environment. Any manipulation of the tissue (e.g. for the isolation or enrichment of certain structures) is noticed by the cells and leads to an adapted gene expression. This state is no longer native and therefore no longer to be regarded as representative.

The human skin has hair down to the lips, the palms of the hands and the soles of the feet. The inconspicuous vellus hairs do not only differ macroscopically e.g. of the cosmetically relevant hair on the head. The hair follicles but also the hair itself can be differentiated microscopically. Which biochemical and molecular biological mechanisms underlie these differences is largely unknown.

A relevant characteristic of hair and its follicles is that with age the cells lose their ability to maintain the organ's homeostasis. For example, the number of hair follicles per unit area decreases over time. The structure of the hair also changes, for example by reducing the hair diameter. With age, certain cells of the hair follicle often no longer produce hair pigments - the hair gray. Which molecular mechanisms underlie this development has so far been largely unclear.

Effective cosmetic or pharmaceutical hair products should have a positive effect on the broadest possible range of molecular processes in the hair follicle. So far, however, only a few molecular reaction mechanisms have been described in the hair follicle, which can thus serve as a target for cosmetic hair products.

Each cell type of the skin and its appendages express approximately 15,000 different genes and synthesize a corresponding number of proteins from them. So far, which genes play a role in hair follicles is largely unclear.

The skin consists of several different cell types (e.g. fibroblasts, keratinocytes in different differentiation states, melanocytes, Merkel cells, Langerhans cells, a large number of different cells of the hair follicle or other skin appendages), so that the complexity of genes expressed in the skin is very great. Up to now it has not been possible to identify the genes associated with the hair follicle from this complexity and which can serve as molecular markers of hair biology. To make matters worse, mRNA molecules are present in the cell in concentrations between a few and several hundred copies. The weakly expressed genes have not been available to previous analysis techniques or have been very difficult to access, but can certainly play a decisive role in the hair follicle.

The transcriptome, i.e. the entirety of all transcribed genes of the human hair follicle, has not been described to date.

Transcriptome analyzes of the skin using various methods, including SAGE ™ analysis, are state of the art. However, isolated keratinocytes (in vitro) or epidermal explants are used, which - as above explained - do not represent models representative of complex skin events.

From DE-A-101 00 127.4-41 by the applicant it is known to subject skin cells to a SAGE ™ analysis in order to characterize the total transcriptome of the skin. DE-A-101 00 121.5-41 by the applicant discloses the determination of markers of stressed or aged skin on the basis of a comparative SAGE ™ analysis between stressed or aged skin and undressed or young skin. However, information on specific markers of hairy skin cannot be found in these publications.

From J Invest Dermatol 2002 Jul; 119 (1): 3-13; "A serial analysis of gene expression in sun-damaged human skin"; Urschitz J. et al .; it is known to determine markers of sun-damaged skin by means of a comparative SAGE ™ analysis of whole-skin explants that are in front of the auricle (sun-damaged) or behind from the auricle (protected from solar radiation), and no knowledge of specific markers on hairy skin can be gained from this publication.

There is therefore a need to identify as many, preferably all, of the genes important for the hair on the skin.

The object of the present invention is to identify as large a part as possible of the genes which are important for the hair on the skin. In addition, methods for determining the homeostasis of hairy skin are to be provided using the identified genes.

This first object is achieved according to the invention by a method (1) for

Identification of the genes which are important for hair on the skin in humans in vitro, characterized in that a) a first mixture of genetically coded factors expressed in hairy human skin, that is to say transcribed and possibly also translated, that is to say of proteins, mRNA molecules or fragments of Proteins or mRNA molecules from hairy human skin, preferably from hairy scalp, b) obtains a second mixture of genetically coded factors expressed in hairless human skin, ie, transcribed and possibly also translated, ie proteins, mRNA molecules or fragments of proteins or mRNA molecules from hairless human skin, preferably from hairless facial skin, and c) subjecting the mixtures obtained in a) and b) to a serial analysis of gene expression (SAGE), thereby identifying the genes that differ in hairy and hairless skin are strongly (differentially) expressed.

In the context of the present invention, “hairless” is also to be understood as meaning the hair with the fine and barely visible vellus hair.

The method according to the invention advantageously makes it possible to understand the complex process of hair formation and the causal relationships between the changes in the hair. Only with this knowledge can new concepts for cosmetic hair products be developed that have an effect on the broad spectrum of gene expression in the hair follicle. The SAGE ™ analysis carried out in the context of the method according to the invention shows for the first time in a very comprehensive manner which genes are expressed in (with the main hair) hairy scalp and which genes are expressed there differently than in skin with vellus hair.

The cosmetic hair products on the market usually exert their effects on the hair shaft (eg hair colorations). Only the studies described here allow an understanding of the complex biological processes in the hair follicle. The identification of suitable markers of the hair follicle thus enables the targeted search for substances or combinations of substances with a broad spectrum of action on gene expression in the hair follicle. However, products of this type could cannot be developed at this time because a large number of hair follicle markers were not yet known.

The technique of "serial analysis of gene expression" (SAGE ™) was used to record the transcriptome of the hairy skin. This technique simultaneously allows the identification and quantification of all genes expressed in the hairy skin. The analysis of the gene expression is also more specific with the quantification mRNA molecules possible (eg Northern blot, RNase protection experiments). However, these techniques can only measure a relatively limited number of genes. In theory, the techniques MPSS (Massive Parallel Signiture Sequencing) or techniques based on differential display could replace the SAGE ™ analysis, but in practice the SAGE ™ technique is faster and more reliable than alternative methods and therefore preferable.

The comparison of the transcriptome of hairy skin with the transcriptome of hairless skin makes it possible to distinguish between genes that are relevant and not relevant for the hair on the skin.

Human skin from healthy female donors was used for SAGE ™ analysis. The SAGE ™ analysis was carried out as described in EP-A-0 761 822 and in Velculescu, VE et al., 1995 Science 270, 484-487. This technique also allows the identification and quantification of the genes expressed in hairy scalp. The comparison of the transcriptome of hairy scalp with the transcriptome of facial skin allows the identification of relevant genes for hairy scalp. These can be genes that are particularly strongly expressed in hairy scalp or also genes that are characterized in that they are only expressed slightly compared to hairless facial skin. The SAGE ™ analysis is a technique with special sensitivity and surprisingly shows even interindividual differences in the gene expression profile. To describe the transcriptome of hairy skin, the comparison to the transcriptome of hairless skin is therefore particularly effective when the analyzed tissue is from an individual come from a donor. Thus, interindividual differences in gene expression are negligible. In the context of plastic surgical operations such as "lower facelifts", hairy tissues accumulate from a patient from the area above the ear and the area behind the ear. At the same time, skin tissue in front of the ear is removed that - at least in female donors - only vellus The analysis of such tissue samples therefore allows the description of the transcriptome of hairy scalp and hairless (or only covered with vellus hair) facial skin with the exclusion of interindividual gene expression differences.

Human skin from a healthy female donor (65 years old) was used for the SAGE ™ analysis. The SAGE ™ analysis was carried out as in EP-A-0 761 822 and at Velculescu, V.E. et al., 1995 Science 270, 484-487. Two SAGE ™ libraries from different localized skin samples from a patient were analyzed. The first sample came from a sampling point above the ear and delivered skin with scalp hair. The second sample came from the same operation, was taken from the ear and provided skin with only vellus hair. For further analysis, both SAGE ™ libraries were standardized to the average number of tags. The two libraries were compared to identify genes with hair-specific regulation. As expected for two libraries of the same tissue type, the tag repertoire of the two skin libraries is largely similar. Despite the similarity of the tissues and the relatively low number of tags, 74 tags show a significantly differential expression with a significance level of p> 0.05.

For hairy skin, an increased number of tags can be shown for genes that are expressed in the hair follicle or in hair appendages. In addition to an increased level of keratin, which is typical of hair and hair follicles, the hairy skin also has, for example, more FGF7, a transcription factor involved in hair development. Dermcidin and cystatin, both involved in the defense against bacterial and viral attacks in the hair shaft, also show a significantly increased expression in the hairy skin. The expression of differentiation-dependent genes of the interfollicular epidermis is in the hairless skin intensified, which is reflected in the results of the investigation. At the top of the differentially expressed genes is keratin 10, a typical marker for the differentiation of stratified epithelia. Keratin 1, the partner of Keratin 10, does not show these differences in expression. The analysis of further differentiation-dependent expressed genes also confirms their increased expression in the hairless skin (Tab. 2). According to the literature, another keratin, keratin 2e, is mainly expressed in hairless skin, which is also observed in the analysis carried out. As expected, the expression of keratins 5, 14 and 15 expressed in the basal layer of the epidermis and in the hair follicles is almost identical in both samples (Tab. 2).

Table 1 lists markers which, according to literature knowledge, are differentially in the hair follicle in the

Compared to the rest of the skin (interfollicular skin). They serve as positive controls for the experiment.

+ H = with hair (skin source above the ear)

-H = without hair (skin source in front of the ear)

Table 2 shows differentiation-dependent genes of the interfollicular epidermis. HF = hair follicle

ORS = Outer Root Sheath (Outer Root Sheath) IRS = Inner Root Sheath (Inner Root Sheath)

+ H = with hair (skin source above the ear)

-H = without hair (skin source in front of the ear)

Further SAGE ™ analyzes of human skin were taken into account to describe and ensure sub-significant differences in gene expression (Tables 7 to 12). It is to be expected, for example, that a hair-specific gene is not only slightly expressed in hairless facial skin, but also in other hairless body skin. The sub-significant differences from the comparison of the transcriptome of hairy scalp with the transcriptome of hairless facial skin were therefore compared with the expression profile of hairless breast skin. For the SAGE ™ analysis of breast skin, breast skin from a healthy female donor (69 years old) used. This confirmed subsignificant differences in gene expression.

Tables 3 to 12 contain a detailed list of the genes determined with the aid of the method according to the invention and differentially expressed in hairy and hairless skin, with indication

• a serial number in column 1,

The tag sequence used in column 2,

The quotient of the determined relative expression frequency in hairy scalp (scalp) and the determined relative expression frequency in hairless facial skin (face) in column 3,

The significance of the values mentioned in column 3 in column 4,

The quotient of the determined relative expression frequency in hairy scalp (scalp) and the determined relative expression frequency in hairless body skin (breast) in column 5,

The significance of the values mentioned in column 5 in column 6,

• the UniGene Accession Number in column 7,

• a brief description of the gene or gene product in column 8 and

• in tables 8, 10 and 12 of the quotient (Scalp / Face) / (Scalp / Breast) in column 9.

The quotient in column 3 indicates the strength of the differential expression, i.e. i.e. by which factor the respective gene is expressed more strongly in hairy scalp (scalp) than in hairless facial skin (face), or vice versa.

The quotient in column 5 indicates the strength of the differential expression, i. i.e. by which factor the respective gene is expressed more strongly in hairy scalp (scalp) than in hairless body skin (breast), or vice versa.

The respective genes or gene products are disclosed in the database of the National Center for Biotechnology Information (NCBI) under their UniGene Accession Number. This database is accessible on the Internet at the following address: http://www.ncbi.nlm.nih.gov/. The genes or gene products are also available at http://www.ncbi.nlm.nih.gov/UniGene/Hs.Home.html or http://www.ncbi.nlm.nih.gov/genome/ guide directly accessible.

Table 3 lists all genes that are at least 10-fold differentially expressed in hairy scalp compared to hairless facial skin with a p-value of p> 0.05 (Signif> 1.3).

Table 4 lists all genes that are expressed differentially in hairy scalp (scalp) compared to hairless facial skin (face) with a p-value of p> 0.05 (Signif> 1, 3) at least 5-fold.

Table 5 lists all genes that are at least 3-fold differentially expressed in hairy scalp compared to hairless facial skin with a p-value of p> 0.05 (Signif> 1, 3).

Table 6 lists all genes that are expressed at least 1.9 times differentially in hairy scalp compared to hairless facial skin with a p-value of p> 0.05 (Signif> 1, 3) ,

Table 7 lists all genes that are expressed differentially in hairy scalp (scalp) compared to hairless facial skin (face) with a p-value of p <0.05 (Signif <1, 3) and that in hairy scalp (scalp) compared to hairless body skin (breast) with a p-value of p> 0.05 (Signif> 1, 3) at least 5-fold differentially expressed. The comparison of the subsignificant scalp / face data with independent SAGE ™ experiments (scalp / breast) confirms the differential gene expression and validates the markers of the hairy skin.

Table 8 lists all the genes that are expressed differentially in hairy scalp (scalp) compared to hairless facial skin (face) with a p-value of p <0.05 (Signif <1, 3) and that in hairy scalp (scalp) compared to hairless body skin (breast) with a p- Value of p <0.05 (Signif <1.3) are expressed at least 5-fold differentially, the expression of which differs by less than a power of ten, ie the quotient (scalp / face) / (scalp / breast) is smaller than 10 or greater than 0.1. The comparison of the subsignificant scalp / face data with independent SAGE ™ experiments (scalp / breast) confirms the differential gene expression and validates the markers of the hairy skin.

Scalp (Scalp) Errr compared to hairless facial skin (Face) with a p-value of p <0.05 (Signif <1, 3) at least 3 times differentially expressed and that in hairy scalp (scalp) compared to hairless body skin (Breast) with a p-value of p> 0.05 (Signif> 1, 3) are expressed at least 3 times differentially. The comparison of the subsignificant scalp / face data with independent SAGE ™ experiments (scalp / breast) confirms the differential gene expression and validates the markers of the hairy skin.

Table 10 lists all genes that are at least 3-fold differentially expressed in hairy scalp compared to hairless facial skin with a p-value of p <0.05 (Signif <1, 3) and that in hairy scalp (scalp) compared to hairless body skin (breast) with a p-value of p <0.05 (Signif <1, 3) at least 3-fold differentially expressed, the expression of which differs by less than a power of ten, that is, the quotient (scalp / face) / (scalp / breast) is less than 10 or greater than 0.1. The comparison of the subsignificant scalp / face data with independent SAGE ™ experiments (scalp / breast) confirms the differential gene expression and validates the markers of the hairy skin.

Table 11 lists all genes that are expressed differentially in hairy scalp (scalp) compared to hairless facial skin (face) with a p-value of p <0.05 (Signif <1.3) at least 1.9-fold and which are expressed differentially in hairy scalp (scalp) compared to hairless body skin (breast) with a p-value of p> 0.05 (Signif> 1, 3) at least 1.9-fold. Comparison of subsignificant scalp / face data with independent SAGE ™ experiments (scalp / breast) confirm the differential gene expression and validate the markers of the hairy skin.

Table 12 lists all genes that are expressed at least 1.9 times differentially in hairy scalp compared to hairless facial skin with a p-value of p <0.05 (Signif <1, 3) and in hairy scalp (scalp) compared to hairless body skin (Breast) with a p-VäTϋe of p <0.05 (signif <1, 3) at least T, 9-fan-shaped red differentially ^{"exf5rirniert} whose expression is less as a power of ten, ie the quotient (Scalp / Face) / (Scalp / Breast) is less than 10 or greater than 0.1. The comparison of the subsignificant Scalp / Face data with independent SAGE ™ experiments (Scalp / Breast) confirms the differential gene expression and validates the markers of the hairy skin.

The second object on which YQ - Jjßg ---- d-aD invention is based is achieved according to the invention by a method (2) for determining the homeostasis of hairy skin in humans, in particular women, in vitro, which is characterized in that a ) a mixture of proteins, mRNA molecules or fragments of proteins or mRNA molecules from hairy human skin is obtained, b) the mixture obtained for the presence and optionally the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA -Molecules examined, which are identified by means of serial analysis of gene expression (SAGE) as differentially expressed in hairy and hairless skin, c) the test results from b) are compared with the expression patterns identified by means of serial analysis of gene expression (SAGE) and d) that in b ) investigated mixture of healthy or hairy skin in homeostasis if it predominantly contains proteins , mRNA molecules or fragments of proteins or mRNA molecules that are more strongly expressed in hairy skin than in hairless skin, or the mixture examined in b) of diseased or hairy skin in disturbed homeostasis, if it predominantly contains proteins, mRNA -Molecules or fragments of Contains proteins or mRNA molecules that are expressed more in hairless skin than in hairy skin.

Diseases or disorders of the homeostasis of hairy skin are, for example: pili torti (torsion hair, twisted hair), monilethrix (spindle hair), wool hair (curly hair), hair shaft changes with breaks [Trichorrhexis nodosa, Trichorrhexis invaginata, Trichoschisis, Trichoptilosis for metabolic disorders, pili recurväti, roller hair, changes in hair color [heterochromia, albinism, poliosis (acquired hereditary pigmentlessness of the hair), canitis (physiological graying), hypertrichoses, hirsutism, alopecia (irreversible alopecia: e.g. androgenetic alopecia of men and women ); Reversible alopecia .: e.g. symptomatic diffuse alopecia due to infections, former noxes and medicines, hormonal disorders, diseases, etc.) and alopezia areata.

The mixture in step a) of the method according to the invention for determining the homeostasis of hairy skin can be obtained from whole skin samples, hairy skin equivalents, isolated hair follicles, hair follicle equivalents or cells of hairy skin.

In step b) of the method for determining the homeostasis of hairy skin, it may be sufficient to examine the mixture obtained for the presence of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which can be determined by means of serial analysis of the gene expression ( SAGE) can be identified as differentially expressed in hairy and hairless skin if they are expressed exclusively in hairy or hairless skin only. In all other cases, the amount of differentially expressed molecules must also be examined in step b). that is, expression must be quantified.

In step d) of the method for determining the homeostasis of hairy skin, the mixture examined in b) of healthy hairy skin is assigned if it is predominantly proteins, mRNA molecules or fragments of proteins or Contains mRNA molecules that are more strongly expressed in hairy skin than in hairless skin, ie that the mixture contains either more different compounds typically expressed in hairy skin than those that are typically expressed in hairless skin (qualitative differentiation), or more Contains copies of compounds typically expressed in hairy skin than are typically present in hairless skin (quantitative differentiation). The assignment to diseased or ^" 1rr ^ e ^ oTteT ^~ Hömeö ^~ stäase hairy skin is followed in a complementary manner.

A preferred embodiment of the method according to the invention for determining the homeostasis of hairy skin is characterized in that in step b) the mixture obtained is examined for the presence and optionally the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules , which are defined in Tables 11 and 12 in column 7 by their UniGene Accession Number, in step c) compares the test results from b) with the expression quotients given in Tables 11 and 12 in column 3 and column 5 and in step d) the mixture examined in b) is assigned to healthy hairy skin if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed in healthy hairy skin at least 1.9 times as strongly as in hairless skin , or assigns the mixture examined in b) to diseased or hairy skin in disturbed homeostasis, if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are expressed at least 1.9 times as strongly in hairless skin as in hairy skin.

Another preferred embodiment of the inventive method for

Determination of the homeostasis of hairy skin is characterized in that in step b) the mixture obtained is checked for the presence and, if appropriate, the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are defined in Tables 9 and 10 in column 7 by their UniGene Accession Number, in step c) the test results from b) with those in Tables 9 and 10 in column 3 and column 5 given expression quotient and in step d) assigns the mixture of healthy hairy skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are at least 3 times in healthy hairy skin barren ^"the ih ^~ b) l tersüclτfe mixture diseased or disturbed homeostasis in-hairy skin allocates when it contains mainly proteins, mRNA molecules or fragments of proteins or mRNA molecules that fold in hairless skin at least 3 so highly expressed become like in hairy skin.

A further preferred embodiment of the method according to the invention for determining the homeostasis of hairy skin is characterized in that in step b) the mixture obtained is checked for the presence and optionally the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules examined, which are defined in Tables 7 and 8 in column 7 by their UniGene Accession Number, in step c) compares the test results from b) with the expression quotients given in Tables 7 and 8 in column 3 and column 5 and in Step d) assigns the mixture of healthy hairy skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least 5 times as strongly in healthy hairy skin as in hairless skin, or assigns the mixture examined in b) to diseased or hairy skin in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least 5 times as strongly in hairless skin as in hairy skin. A particularly preferred embodiment of the method according to the invention for determining the homeostasis of hairy skin is characterized in that in step b) the mixture obtained is checked for the presence and optionally the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules examined, which are defined in Table 6 in column 7 by their UniGene Accession Number, in step c) the ^~ OntersuclτüTrgsel ^ eΕ ^ is ^ eäϊ sb) ^~ rnit with ^" deh ^{" in} table 6 ^~ in ^" S ^' column 3 and column 5 expressed expression quotient and in step d) assigns the mixture of healthy hairy skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are at least 1.9 times as good in healthy hairy skin are strongly expressed as in hairless skin, or the mixture examined in b) of sick or hairy in disturbed homeostasis contains proteins or mRNA molecules that are expressed at least 1.9 times as strongly in hairless skin as in hairy skin.

Another particularly preferred embodiment of the invention

The method for determining the homeostasis of hairy skin is characterized in that in step b) the mixture obtained is examined for the presence and, if appropriate, the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which are shown in Table 5 defined in column 7 by their UniGene accession number, in step c) comparing the test results from b) with the expression quotients given in table 5 in column 3 and column 5 and in step d) the mixture of healthy hairy skin examined in b) assigned if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least 3 times as strongly in healthy hairy skin as in hairless skin, or which was examined in b)

Mixture of diseased or hairy skin in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of Contains proteins or mRNA molecules that are expressed at least 3 times as strongly in hairless skin as in hairy skin.

A further particularly preferred embodiment of the method for determining the homeostasis of the scalp is characterized in that in step b) the mixture obtained in the presence and optionally iie ^_ M ^"ertge ^ σn ^_ ^min" de ^"steπs ^~ a ^_ of the proteins ; mRNA molecules or fragments of proteins or mRNA molecules examined, which are defined in Table 4 in column 7 by their UniGene Accession Number, in step c) the test results from b) with those in Table 4 in column 3 and column 5 indicated expression quotient and in step d) assigns the mixture of healthy hairy skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least 5 times as strongly in healthy hairy skin as in hairless skin, or the mixture examined in b) of diseased or hairy skin in disturbed homeostasis arranges if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are expressed at least 5 times as strongly in hairless skin as in hairy skin.

A very particularly preferred embodiment of the invention

A method for determining the homeostasis of hairy skin is characterized in that in step b) the mixture obtained is examined for the presence and, if appropriate, the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which are shown in Table 3 defined in column 7 by their UniGene accession number, in step c) comparing the test results from b) with the expression quotients given in table 3 in column 3 and column 5 and in step d) the mixture of healthy hairy skin examined in b) assigned if it contains predominantly proteins, mRNA molecules or fragments of proteins or mRNA molecules that are at least 10- in healthy hairy skin are expressed as strongly as in hairless skin, or assigns the mixture examined in b) to diseased or hairy skin in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are in hairless skin expressed at least 10 times as strongly as in hairy skin.

The condition of the skin can also be described by quantifying a number of markers (expression products of the genes which are important for hairy skin), which must then be active in a characteristic ratio to one another in order to represent healthy (inostasis) hairy skin, or must be active in a characteristic ratio different from this in order to represent diseased skin (which is in disturbed homeostasis).

Another object of the present invention is therefore a method j3) for determining the homeostasis of hairy skin in humans, in particular women, in vitro, which is characterized in that a) a mixture of proteins, mRNA molecules or fragments of proteins or mRNA molecules from hairy human skin, b) quantified in the mixture obtained at least two of the proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are identified by method (1) as being important for hairy skin, c) the Expression ratios of the at least two proteins, mRNA molecules or fragments of proteins or mRNA molecules to one another are determined and the expression quotient is formed, d) comparing the expression ratios from c) with the expression ratios which are typically hairy or in for the molecules quantified in b) hairless skin, especially with the expression ratios, d ie from tables 3 to 12, columns 3 and 5, respectively, and e) assigns the mixture obtained in a) to healthy (in homeostasis) hairy skin if the expression ratios of the examined skin correspond to the expression ratios in hairy skin, or that in a) obtained mixture of diseased (in disturbed homeostasis) skin assigned if the expression ratios of the examined skin correspond to the expression ratios of hairless skin.

In step a) of the method according to the invention for determining the homeostasis of hairy skin, the mixture is preferably obtained from a skin sample, in particular from a whole skin sample or from an epidermis sample. Here, the ^~~ Völlh ütprbbe ^" Cimfässehdere ^™ opens up possibilities for comparison ^~ with the SAGE libraries, which are also obtained from whole skin. The epidermis sample, on the other hand, is easier to obtain, for example by applying an adhesive tape to the skin and tearing it off, as described in WO 00/10579 , to which reference is hereby made in full.

In a further embodiment of the method according to the invention for determining the homeostasis of hairy skin, the mixture is obtained in step a) by means of microdialysis. The technique of microdialysis is described, for example, in "Microdialysis: A method for measurement of local tissue metabolism", Nielsen PS, Winge K, Petersen LM; Ugeskr Laeger 1999 Mar 22 161: 12 1735-8; and in "Cutaneous microdialysis for human in vivo dermal absorption studies ", Anderson, C. et al. ; Drugs Pharm. Sci., 1998, 91, 231-244; and also described on the Internet at http://www.microdialysis.se/techniqu.htm, to which reference is hereby made in full.

When using microdialysis, a probe is typically inserted into the skin and the probe is slowly rinsed with a suitable carrier solution. After the acute reactions have subsided after the puncture, the microdialysis provides proteins, mRNA molecules or fragments of proteins or mRNA molecules which occur in the extracellular space and which can then be isolated and analyzed in vitro, for example by fractionation of the carrier liquid. Microdialysis is less invasive than taking a full skin sample; however, it is disadvantageously limited to the extraction of compounds occurring in the extracellular space. A further preferred embodiment of the method according to the invention for determining the homeostasis of hairy skin is characterized in that in step b) in method (2) the examination for the presence and optionally the amount of at least one of the proteins or protein fragments; or in method (3) the quantification of at least two proteins or protein fragments is carried out by means of a method which is selected from ii. Affinity Chromatography iii. Protein-protein complexation in solution iv. Mass spectrometry, especially matrix assisted laser desorption ionization (MALDI) and especially v. Use of protein chips, or by means of suitable combinations of these methods.

These methods, which can be used according to the invention, are described in the review article by Akhilesh Pandey and Matthias Mann: "Proteomics to study genes and genomes", Nature, Volume 405, Number 6788, 837 - 846 (2000), and the references given therein, which are hereby fully described Scope is referred to.

2D gel electrophoresis is described, for example, in L.D. Adams, Two-dimensional Gel Electrophoresis using the Isodalt System or in L.D. Adams & S.R. Gallagher, Two-dimensional Gel Electrophoresis using the O'Farrell System; both in Current Protocols in Molecular Biology (1997, Eds. F.M. Ausubel et al.), Unit 10.3.1 - 10.4.13; or in 2-D electrophoresis manual; T. Berkelman, T. Senstedt; Amersham Pharmacia Biotech, 1998 (order no. 80-6429-60).

The mass spectrometric characterization of the proteins or protein fragments is carried out in a manner known to those skilled in the art, for example as described in the following references: Methods in Molecular Biology, 1999; Vol 112; 2-D Proteome Analysis Protocols; Editor: AJ Link; Humana Press; Totowa; New Jersey. In particular: Courchesne, PL and Patterson, SD; Pp. 487-512.

Carr, S.A. and Annan, R. S .; 1997; in: Current Protocols in Molecular Biology; Editor: Ausubel, F. M. et al .; John Wiley and Sons, Inc. 10.2.1-10.21.27.

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Determination of the homeostasis of hairy skin is characterized in that in step b) in method (2) the examination for the presence and, if appropriate, the amount of at least one of the mRNA molecules or mRNA molecule fragments; or in method (3) the quantification of at least two mRNA molecules or mRNA molecule fragments is carried out by means of a method which is selected from Northern blnts.

Reverse transcriptase polymerase chain reaction (RT-PCR), RNase protection experiments, iv. Dot blots, v. cDNA sequencing, vi. Clone hybridization, vii. Differential display, viii. Subtractive hybridization, ix. cDNA fragment fingerprinting, x. Total Gene Expression Analysis (TOGA), xi. Serial analysis of gene expression (SAGE), xii. Massively Parallel Signature Sequencing (MPSS ^® ) and especially xiii. Use of nucleic acid chips, or by means of suitable combinations of these methods.

These methods according to the invention can be used in the review articles by Akhilesh Pandey and Matthias Mann: "Proteomics to study genes and genomes", Nature, Volume 405, Number 6788, 837 - 846 (2000), and "Genomics, gene expression and DNA arrays", Nature, Volume 405, Number 6788, 827-836 (2000), and the references given there, to which reference is hereby made in full.

The TOGA method is described in "J. Gregor Sutcliffe et al, TOGA: An automated parsing technology for analyzing expression of nearly all genes, Proceedings of the National Academy of Sciences of the United States of America (PNAS), Vol. 97, No . 5, pp. 1976-1981 (2000) ", to which reference is hereby made in full. full reference is made.

However, according to the invention, other methods known to the person skilled in the art can also be used to examine for the presence and, if appropriate, the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules.

Another preferred embodiment of the method according to the invention for determining the homeostasis of hairy skin is characterized in that in step b) the presence and optionally the amount of 1 to about 5000, preferably 1 to about 1000, in particular about 10 to about 500, preferably about 10 to about 250, particularly preferably about 10 to about 100 and very particularly preferably about 10 to about 50 of the proteins, mRNA molecules or fragments of proteins or mRNA molecules investigated by their in Tables 3 to 12 in column 7 UniGene Accession Number can be defined.

The present invention further provides a test kit for determining the homeostasis of hairy skin in humans in vitro, comprising means for carrying out the method according to the invention for determining the homeostasis of hairy skin.

Another object of the present invention is a biochip for determining the homeostasis of hairy skin in humans in vitro, comprising i. a solid, ie rigid or flexible support and ii. on this immobilized probes which are capable of specific binding to at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which are defined in Tables 3 to 12 in column 7 by their UniGene Accession Number.

A biochip which is particularly preferred according to the invention comprises probes which are selected from those which specifically bind to at least one

Molecules are capable, which are defined in Tables 3 to 6 in column 1 by the following serial number: 2, 4, 9, 12, 14, 16, 22, 25, 29, 31, 35, 36, 38, 39, 40 , 42, 43, 44, 46, 59, 62, 63, 65, 67, 68, 69, 74.

A BioChip is a miniaturized functional element with molecules immobilized on a surface, in particular biomolecules, which can serve as specific interaction partners.

The structure of these functional elements often has rows and columns; one then speaks of chip "arrays". Since thousands of biological or biochemical functional elements can be arranged on a chip, they usually have to be manufactured using microtechnical methods. The following are particularly suitable as biological and biochemical functional elements: DNA, RNA, PNA (in the case of nucleic acids and their chemical derivatives, for example, single strands, triplex structures or combinations thereof), saccharides, peptides, proteins (for example antibodies) , Antigens, receptors) and derivatives of combinatorial chemistry (e.g. organic molecules). In general, BioChips have a 2D base area for coating with biologically or biochemically functional materials. The base surfaces can also be formed, for example, by walls of one or more capillaries or by channels.

The prior art can e.g. For example, reference is made to the following publications: Nature Genetics, Vol. 21, Supplement (overall), Jan. 1999 (BioChips); Nature Biotechnology, Vol. 16, pp. 981-983, Oct. 1998 (BioChips); Trends in Biotechnology, Vol. 16, pp. 301-306, Jul. 1998 (BioChips) as well as the review articles by Akhilesh Pandey and Matthias Mann already mentioned: "Proteomics to study genes and genomes", Nature, Volume 405, Number 6788, 837-846 (2000), and "Genomics, gene expression and DNA arrays", Nature, Volume 405, Number 6788, 827-836 (2000), and the references given there, to which reference is hereby made in full.

The books "DNA Microarrays: A Practical Approach"^" (Editor: MäTfTSchena, ^" 19997 ^" OxföT OFπve7sity ^~ Pf ess) ^~ and ^~ M icröäTräy ^~ Biöchip Technology" (Editor: Mark Schena, 2000, Eaton Publishing), to which reference is hereby made in full.

The DNA chip technology which is particularly preferred in the context of the present invention is based on the ability of nucleic acids to enter into complementary base pairings. This technical principle, known as hybridization, has been used in Southern blot and Northern blot analysis for years. Compared to these conventional methods, in which only a few genes are analyzed, DNA chip technology allows a few hundred to several tens of thousands of genes to be examined in parallel. A DNA chip essentially consists of a carrier material (e.g. glass or plastic) on which single-stranded, gene-specific probes are immobilized in a high density at a defined location (spot). The technique of probe application and the chemistry of probe immobilization are considered problematic.

According to the current state of the art, several ways of immobilizing probes are realized:

E. M. Southern (EM Southern et al. (1992), Nucleic Acid Research 20, 1679-1684 and EM Southern et al. (1997), Nucleic Acid Research 25, 1155-1161) describes the preparation of oligonucleotide arrangements by direct synthesis on a glass surface, which was derivatized with 3-glycidoxypropyltrimethoxysilane and then with a glycol.

A similar process realizes the in situ synthesis of oligonucleotides by means of a photosensitive, combinatorial chemistry, which with photolithographic Techniques can be compared (Pease, AC et al. (1994), Proc. NatI Acad Sei USA 91, 5022-5026).

In addition to these techniques based on the s / fu synthesis of oligonucleotides, DNA molecules already present can also be bound to surfaces of support material.

PO Brown (DeRlsi et al7 ^~ (T997), ^~ Science 2787 ^"6BO ^ ^686)" bes ^ ^~ lτreirJt clie immobilization of DNA to polylysine coated glass surfaces. The publication by LM Smith (Guo, Z. et al. (1994), Nucleic Acid Research 22, 5456-5465) discloses a similar process: oligonucleotides which carry a 5'-terminal amino group can be bound to a glass surface which was treated with 3-aminopropyltrimethoxysilane and then with 1,4-phenyldiisothioeyanate.

The DNA probes can be applied to a carrier using a so-called “pin spotter”. For this purpose, thin metal needles, for example with a diameter of 250 μm, are immersed in probe solutions and then transfer the attached sample material with defined volumes to the carrier material of the DNA Crisps.

However, the probe is preferably applied by means of a piezocontrolled nanodispenser, which, like an inkjet printer, applies probe solutions with a volume of 100 picoliters to the surface of the carrier material without contact.

The probes are immobilized, for example, as described in EP-A-0 965 647: The generation of DNA probes is carried out by means of PCR using a sequence-specific pair of primers, a primer at the 5 'end being modified and a linker with a free one Amino group carries. This ensures that a defined strand of the PCR products can be bound to a glass surface which has been treated with 3-aminopropyltrimethoxysilane and then with 1,4-phenyldiisothiocyanate. The Gene-specific PCR products should ideally have a defined nucleic acid sequence with a length of 200-400 bp and contain non-redundant sequences. After the immobilization of the PCR products via the derivatized primer, the counter strand of the PCR product is removed by incubation at 96 ° C. for 10 minutes.

In an application typical for DNA chips, mRNA from two to NeTgleicheTTäeTrZellpopulä ^ mRNÄs are converted into cDNA by means of ^" reverse transcription using, for example, fluorescence-labeled nucleotides. The samples to be compared are labeled with, for example, red or green fluorescent nucleotides. The cDNAs are labeled then hybridized with the gene probes immobilized on the DNA chip and subsequently quantified the bound fluorescence.

The analysis chips mentioned in DE-A-100 28 257.1-52 and in DE-A-101 02 063.5-52 are particularly preferred for the production of small (up to about 500 probes) biochips. These analysis chips have an electrically addressable structure which allows the samples to be electrofocused. This advantageously makes it possible to focus and immobilize samples regardless of their viscosity with the aid of electrodes at defined points on a grid of points (arrays). Due to the focusing ability, the local concentration of the samples is increased and thus a higher specificity. During the analysis itself, it is possible to address the test material to the individual positions in the array. In this way, any investigated information can potentially be tracked down with the highest possible sensitivity. Cross-contamination from neighboring spots is almost impossible.

The biochip according to the invention preferably comprises 1 to approximately 5000, preferably 1 to approximately 1000, in particular approximately 10 to approximately 500, preferably approximately 10 to approximately 250, particularly preferably approximately 10 to approximately 100 and very particularly preferably approximately 10 to approximately 50 different probes. The probes, which differ from one another, can each be present in duplicate on the chip. The biochip according to the invention preferably comprises nucleic acid probes, in particular RNA or PNA probes, particularly preferably DNA probes. The nucleic acid probes preferably have a length of approximately 10 to approximately 1000, in particular approximately 10 to approximately 800, preferably approximately 100 to approximately 600, particularly preferably approximately 200 to approximately 400 nucleotides.

Another preferred form of the biochip according to the invention comprises peptide or protein probes, in particular antibodies.

Another object of the present invention is the use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are defined in Tables 3 to 12 in column 7 by their UniGene accession number, as markers of hairy skin in humans ,

In this regard, some are those listed in Tables 3 through 12

Genes or gene products of particular interest, for example the protein

CDT6, whose role as a hair cycle marker is particularly surprising since it would have been more likely that VEGF or angiopoietin could be used as a marker.

The same applies to the proteins of the 14-3-3 family. Although these are ubiquitously expressed, it was not possible to predict which isoforms would play a role in

Play hair cycle, what expression pattern these isoforms will have.

Among the overrepresented groups, the DPPIV family and the DNA helicases are particularly noticeable. For both gene families, a role in the hair cycle was also not foreseeable by the expert.

The genes of melanin biosynthesis should also be mentioned here. That the genes of

Melanin biosynthesis in catagen hair follicles is more strongly expressed than in anagen is unexpected and surprising.

Another object of the present invention is a test method for demonstrating the effectiveness of cosmetic or pharmaceutical active ingredients against diseases or impairments of the homeostasis of hairy skin in vitro, characterized in that a) the skin status of hairy skin by an inventive method for determining the homeostasis of hairy skin, or by means of a test kit according to the invention for determining the homeostasis of hairy skin, or by means of a biochips according to the invention determines ^"&) a drug counter --i! rlä ι ^θngeτr" ^ eT ^~ Beeinträchtigαrrgen ^{^} homeostasis hairy skin once or more is applied to the hairy skin, c) was again the skin status hairy skin by an inventive method for determining the homeostasis of hairy skin, or using a test kit according to the invention for determining the homeostasis of hairy skin, or using a biochip according to the invention and d) determining the effectiveness of the active ingredient by comparing the results from a) and c).

The test method according to the invention can be carried out with whole skin samples, hairy skin equivalents, isolated hair follicles, hair follicle equivalents or cells with hairy skin.

Another object of the present invention is a test kit for demonstrating the effectiveness of cosmetic or pharmaceutical active substances against diseases or impairments of the homeostasis of hairy skin, comprising means for carrying out the test method according to the invention.

Another object of the present invention is the use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are defined in Tables 3 to 12 in column 7 by their UniGene Accession Number, for the detection of the effectiveness of cosmetic or pharmaceutical active substances against diseases or impairments of the homeostasis of hairy skin. Another object of the present invention is a screening method for the identification of cosmetic or pharmaceutical active substances against diseases or impairments of the homeostasis of hairy skin in vitro, which is characterized in that a) the skin status of hairy skin by an inventive method for determining the homeostasis hairy skin, or by a ^ rfindungsgemäßen- -estrKits-for-determination ^_ ^_ derΗomeostase behaarteτ ^"skin, or determined by means of a biochip according to the invention, b) a potential drug for diseases or disturbances of homeostasis the scalp once or more is applied to the skin, c) the skin status of hairy skin is determined by a method according to the invention for determining the homeostasis of hairy skin, or by means of a test kit according to the invention for determining the homeostasis of hairy skin, or by means of a biochip according to the invention, and d) effective active ingredient ffe determined by comparing the results from a) and c).

Another object of the present invention is the use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are defined in Tables 3 to 12 in column 7 by their UniGene Accession Number, for the identification of cosmetic or pharmaceutical Active substances against diseases or impairments of the homeostasis of hairy skin.

Another object of the present invention is a method for producing a cosmetic or pharmaceutical preparation against diseases or impairments of the homeostasis of hairy skin, characterized in that a) active ingredients using the screening method according to the invention, or the use for the identification of cosmetic or active pharmaceutical ingredients against diseases or

Impairments in the homeostasis of hairy skin are determined and active ingredients found to be effective are mixed with cosmetically and pharmacologically suitable and compatible carriers.

tables:

Table 1:

Table 2:

Table 3:

Table 4:

Table 5:

Table 6:

Table 7:

Table 8:

Table 9:

Table 10:

tables:

Table 12:

Claims

claims

1. A method for identifying the genes important for hair on the skin in humans in vitro, characterized in that a) a first mixture of expressed in hairy human skin, d. H. transcribed and optionally also translated genetically coded factors, ie proteins, mRNA molecules or fragments of proteins or mRNA molecules from hairy human skin, b) a second mixture of expressed in hairless human skin, d. H. transcribed and possibly also translated genetically coded factors, i.e. proteins, mRNA molecules or fragments of proteins or mRNA molecules from hairless human skin, and c) subjecting the mixtures obtained in a) and b) to a serial analysis of gene expression (SAGE) , and thereby identifies the genes that are expressed to different extents in hairy and hairless skin.

2. The method according to claim 1, characterized in that the hairy human skin is hairy scalp and the hairless human skin is hairless facial skin.

3. A method for determining the homeostasis of hairy skin in humans in vitro, characterized in that a) a mixture of proteins, mRNA molecules or fragments of proteins or mRNA molecules from hairy human skin is obtained, b) the mixture obtained on the Existence and, if appropriate, the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which are identified by differential analysis of gene expression (SAGE) as being differentially expressed in hairy and hairless skin, c) the test results from b ) with the expression patterns identified by means of serial analysis of gene expression (SAGE) and d) assigns the mixture examined in b) to healthy or hairy skin located in homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed more strongly in hairy skin than in hairless skin, or that in b) examined mixture of diseased or hairy skin located in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed more in hairless skin than in hairy skin.

4. The method according to claim 3, characterized in that a) in step b) the mixture obtained for the presence and optionally the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules that are examined in the Tables 11 and 12 in column 7 are defined by their UniGene accession number, b) in step c) the test results from b) are compared with the expression quotients given in tables 11 and 12 in column 3 and column 5 and c) in step d) the mixture examined in b) is assigned to healthy hairy skin if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed in healthy hairy skin at least 1.9 times as strongly as in hairless skin , or the mixture examined in b) of diseased or hairy skin in disturbed homeostasis, if it predominantly contains proteins, mRNA molecules or fragments of proteins or contains mRNA molecules that are expressed in hairless skin at least 1.9 times as strongly as in hairy skin.

5. The method according to claim 3, characterized in that a) in step b) the mixture obtained for the presence and optionally the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules examined, the in tables 9 and 10 in column 7 are defined by their UniGene Accession Number, b) in step c) the test results from b) are compared with the expression quotients given in tables 9 and 10 in columns 3 and 5 and c) in step d) assigns the mixture examined in b) to healthy hairy skin if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least three times as strongly in healthy hairy skin as in hairless skin , or assigns the mixture examined in b) to diseased or hairy skin in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least 3 times as strongly in hairless skin as in hairy skin.

6. The method according to claim 3, characterized in that a) in step b) the mixture obtained for the presence and optionally the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules that are examined in the Tables 7 and 8 in column 7 are defined by their UniGene accession number, b) in step c) the test results from b) are compared with the expression quotients given in tables 7 and 8 in column 3 and column 5 and c) in step d) assigns the mixture examined in b) to healthy hairy skin if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed in healthy hairy skin at least 5 times as strongly as in hairless skin, or assigns the mixture examined in b) to diseased or hairy skin in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mR Contains NA molecules that are expressed at least 5 times as strongly in hairless skin as in hairy skin.

7. The method according to claim 3, characterized in that a) in step b) the mixture obtained is examined for the presence and optionally the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are in table 6 are defined in column 7 by their UniGene accession number, b) in step c) the test results from b) are compared with the expression quotients given in table 6 in column 3 and column 5 and c) in step d) that in b) The examined mixture is assigned to healthy hairy skin if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are expressed in healthy hairy skin at least 1.9 times as strongly as in hairless skin, or that in b) investigated mixture of diseased or hairy skin in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules lt, which are in hairless skin at least 1.9 times as strongly expressed in haired skin.

8. The method according to claim 3, characterized in that a) in step b) the mixture obtained is examined for the presence and optionally the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are in table 5 are defined in column 7 by their UniGene accession number, b) in step c) the test results from b) are compared with the expression quotients given in table 5 in column 3 and column 5 and c) in step d) that in b) The examined mixture is assigned to healthy hairy skin if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed in healthy hairy skin at least three times as strongly as in hairless skin, or the mixture examined in b) assigns to sick or hairy skin in disturbed homeostasis if it contains predominantly proteins, mRNA Contains molecules or fragments of proteins or mRNA molecules that are expressed at least 3 times as strongly in hairless skin as in hairy skin.

9. The method according to claim 3, characterized in that a) in step b) the mixture obtained is examined for the presence and optionally the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are in table 4 are defined in column 7 by their UniGene accession number, b) in step c) the test results from b) are compared with the expression quotients given in table 4 in column 3 and column 5 and c) in step d) that in b) The examined mixture is assigned to healthy hairy skin if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed in healthy hairy skin at least 5 times as strongly as in hairless skin, or the mixture examined in b) assigned to hairy skin or in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least 5 times as strongly in hairless skin as in hairy skin.

10. The method according to claim 3, characterized in that a) in step b) the mixture obtained is examined for the presence and optionally the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are in table 3 are defined in column 7 by their UniGene accession number, b) in step c) the test results from b) are compared with the expression quotients given in table 3 in column 3 and column 5 and c) in step d) that in b) investigated mixture assigned to healthy hairy skin if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are found in healthy hairy skin are expressed at least 10 times as strongly as in hairless skin, or the mixture examined in b) of diseased or hairy skin located in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which expressed at least 10 times as much in hairless skin as in hairy skin.

11. A method for determining the homeostasis of hairy skin in humans, in particular women, in vitro, which is characterized in that a) a mixture of proteins, mRNA molecules or fragments of proteins or mRNA molecules is obtained from hairy human skin, b) in the mixture obtained, at least two of the proteins, mRNA molecules or fragments of proteins or mRNA molecules are quantified, which are identified as being important for hairy skin by means of a method according to claim 1, c) the expression ratios of the at least two proteins, mRNA Molecules or fragments of proteins or mRNA molecules are determined with respect to one another and form the expression quotient, d) compares the expression ratios from c) with the expression ratios that are typically present in the hairy or hairless skin for the molecules quantified in b), in particular with the expression ratios , which are derived from Tables 3 to 12, Columns 3 or 5, and e) assigns the mixture of healthy (in homeostasis) hairy skin obtained in a) if the expression ratios of the examined skin correspond to the expression ratios in hairy skin, or the mixture obtained in a) sick (in disturbed homeostasis) ) Skin assigned if the expression ratios of the examined skin correspond to the expression ratios of hairless skin.

12. The method according to any one of claims 3 to 10, characterized in that the examination in step b) for the presence and optionally perform the amount of at least one of the proteins or protein fragments by a method selected from i. One- or two-dimensional gel electrophoresis ii. Affinity Chromatography iii. Protein-protein complexation in solution iv. Mass spectrometry, especially matrix assisted laser desorption ionization (MALDI) and especially v. Use of protein chips, or by means of suitable combinations of these methods.

13. The method according to claim 11, characterized in that one carries out the quantification of at least two proteins or protein fragments by means of a method which is selected from i. One- or two-dimensional gel electrophoresis ii. Affinity Chromatography iii. Protein-protein complexation in solution iv. Mass spectrometry, especially matrix assisted laser desorption ionization (MALDI) and especially v. Use of protein chips, or by means of suitable combinations of these methods.

14. The method according to any one of claims 3 to 10 and 12, characterized in that one carries out the investigation in step b) for the presence and optionally the amount of at least one of the mRNA molecules or mRNA molecule fragments by means of a method which is selected under i. Northern Blots, ii. Reverse transcriptase polymerase chain reaction (RT-PCR), iii. RNase protection experiments, iv. Dot blots, v. CDNA sequencing, vi. Clone hybridization, vii. Differential display, viii. Subtractive hybridization, ix. cDNA fragment fingerprinting, x. Total Gene Expression Analysis (TOGA) xi. Serial analysis of gene expression (SAGE), xii. Massively Parallel Signature Sequencing (MPSS ^® ) and especially xiii. Use of nucleic acid chips, or by means of suitable combinations of these methods.

15. The method according to claim 11 or 13, characterized in that one carries out the investigation in step b) the quantification of at least two mRNA molecules or mRNA molecule fragments by means of a method which is selected from Northern blots, reverse transcriptase polymerase chain reaction (RT-PCR ), iii. RNase protection experiments, iv. Dot blots, v. CDNA sequencing, vi. Clone hybridization, vii. Differential display, viii. Subtractive hybridization, ix. cDNA fragment fingerprinting, x. Total Gene Expression Analysis (TOGA) xi. Serial analysis of gene expression (SAGE) and especially xii. Massively Parallel Signature Sequencing (MPSS ^® ) and especially xiii. Use of nucleic acid chips, or by means of suitable combinations of these methods.

16. The method according to any one of claims 3 to 15, characterized in that in step b) the presence and optionally the amount of 1 to about 5000, preferably 1 to about 1000, in particular about 10 to about 500, preferably about 10 to about 250, particularly preferably about 10 to about 100 and very particularly preferably about 10 to about 50 of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which are examined in tables 3 to 12 in column 7 are defined by their UniGene Accession Number.

17. Test kit for determining the homeostasis of hairy skin in humans in vitro, comprising means for performing the method according to one of claims 3 to 16.

18. Biochip for determining the homeostasis of hairy skin in humans in vitro, comprising i. a firm, d. H. rigid or flexible beams and ii. on this immobilized probes which are capable of specific binding to at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which are defined in Tables 3 to 12 in column 7 by their UniGene Accession Number, in particular to those defined in Tables 3 to 6 in column 1 by the following serial number: 2, 4, 9, 12, 14, 16, 22, 25, 29, 31, 35, 36, 38, 39, 40, 42, 43, 44, 46, 59, 62, 63, 65, 67, 68, 69, 74.

19. Biochip according to claim 18, comprising 1 to about 5000, preferably 1 to about 1000, in particular about 10 to about 500, preferably about 10 to about 250, particularly preferably about 10 to about 100 and very particularly preferably about 10 to about 50 of each other different probes.

20. Biochip according to claim 18 or 19, comprising nucleic acid probes, in particular RNA or PNA probes, particularly preferably DNA probes.

21. The biochip according to claim 20, comprising probes with a length of approximately 10 to approximately 1000, in particular approximately 10 to approximately 800, preferably approximately 100 to approximately 600, particularly preferably approximately 200 to approximately 400 nucleotides.

22. Biochip according to claim 18 or 19, comprising peptide or protein probes, in particular antibodies.

23. Use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are defined in Tables 3 to 12 in column 7 by their UniGene accession number, as markers on hairy skin in humans.

24. Test method for proving the effectiveness of cosmetic or pharmaceutical active substances against diseases or impairments of the homeostasis of hairy skin in vitro, characterized in that a) the skin status of hairy skin by a method according to one of claims 3 to 15, or by means of a test Kits according to claim 17, or by means of a biochip according to one of claims 18 to 22, b) applying an active ingredient against diseases or impairments of the homeostasis of hairy skin to the hairy skin one or more times, c) again the skin status of hairy skin by a method determined according to one of claims 3 to 15, or by means of a test kit according to claim 17, or by means of a biochip according to one of claims 18 to 22, and d) the effectiveness of the active ingredient by comparing the results from a) and c) determined.

25. Test kit for proving the effectiveness of cosmetic or pharmaceutical active substances against diseases or impairments of the homeostasis of hairy skin, comprising means for carrying out the test method according to claim 24.

26. Use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are defined in Tables 3 to 12 in column 7 by their UniGene Accession Number, for demonstrating the effectiveness of cosmetic or pharmaceutical active substances against diseases or Impairment of hairy skin homeostasis.

27. Screening method for identifying cosmetic or pharmaceutical active substances against diseases or impairments of the homeostasis of hairy skin in vitro, which is characterized in that a) the skin status of hairy skin by a method according to any one of claims 3 to 15, or by means of a Test kits according to claim 17, or by means of a biochip according to one of claims 18 to 22, determines, b) applying a potential active substance against diseases or impairments of the homeostasis of hairy skin to the skin one or more times, c) the skin status of hairy skin by a Method according to one of claims 3 to 15, or by means of a test kit according to claim 17, or by means of a biochip according to one of claims 18 to 22, determines d) active ingredients by comparing the results from a) and c).

28. Use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are defined in Tables 3 to 12 in column 7 by their UniGene Accession Number, for the identification of cosmetic or pharmaceutical active substances against diseases or impairments of the Homeostasis of hairy skin.

29. A process for the preparation of a cosmetic or pharmaceutical preparation against diseases or impairments of the homeostasis of hairy skin, characterized in that a) active ingredients are determined using the screening method according to claim 27, or the use according to claim 28, and b) effective found active ingredients mixed with cosmetically and pharmacologically suitable and compatible carriers.