EP1356106A2

EP1356106A2 - Method for determining skin stress or skin ageing in vitro

Info

Publication number: EP1356106A2
Application number: EP01986926A
Authority: EP
Inventors: Dirk Petersohn; Marcus Conradt; Kay Hofmann
Original assignee: Henkel AG and Co KGaA
Current assignee: Henkel AG and Co KGaA
Priority date: 2001-01-03
Filing date: 2001-12-20
Publication date: 2003-10-29
Also published as: WO2002053773A2; DE10100121A1; US20040142335A1; WO2002053773A3

Abstract

The invention relates to a method for determining skin stress and/or skin ageing in humans or animals in vitro, test kits and biochips for determining skin stress and/or skin ageing, and the use of proteins, mRNA molecules or fragments of proteins or mRNA molecules as skin stress and/or ageing markers. The invention also relates to a test method for demonstrating the effectiveness of cosmetic or pharmaceutical active ingredients against skin stress and/or skin ageing, a screening method for identifying cosmetic or pharmaceutical active ingredients against skin stress and/or skin ageing, and a method for producing a cosmetic and/or pharmaceutical preparation against skin stress and/or skin ageing. The invention further relates to a cosmetic or pharmaceutical preparation against skin stress and/or skin ageing.

Description

Method for determining skin stress or skin aging in vitro

The present invention relates to a method for determining skin stress and / or skin aging in humans or animals in vitro, test kits and biochips for determining skin stress and / or skin suppuration, and the use of proteins, mRNA molecules or fragments of proteins or mRNA molecules as stress and / or aging markers of the skin; furthermore a test method for proving the effectiveness of cosmetic or pharmaceutical active substances against skin stress and / or skin aging as well as a screening method for the identification of cosmetic or pharmaceutical active substances against skin stress and / or skin aging and a method for the production of a cosmetic or pharmaceutical preparation against skin stress and / or or skin aging.

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 comprises around 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, each cell responds to signals from its environment by adapting its metabolism.

For example, the cells of the skin notice the high-energy radiation from the sun and react to it by changing their RNA and protein synthesis performance. Some molecules are increasingly synthesized after a stress stimulus (e.g. sunlight) (e.g. MMP-1), others are produced to a lesser extent (e.g. collagen αι (I)). Furthermore, no significant change will take place in a large number of the synthesis processes (for example TIMP-1).

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 and forms the body's interface with the environment. This fact makes it clear that the cells of the skin are particularly exposed to exogenous signals of the environment, physical and chemical nature and therefore regulate their gene expression continuously. To understand skin reactions to exogenous stimuli, the analysis of gene expression in the skin is therefore of crucial importance.

The macroscopic phenomena of aging skin are based on the one hand on intrinsic or chronological aging (skin aging), and on the other hand on extrinsic aging due to environmental stress (skin stress). The ability of living skin cells to react to your environment changes over time - there are aging processes that lead to senescence and ultimately cell death. The visible signs of aged skin are to be understood as an integral of intrinsic and extrinsic aging (e.g. due to sunlight), the events of extrinsic aging accumulating in the skin over a longer period of time.

A crucial characteristic of the skin is that with age the cells lose their ability to maintain the organ's homeostasis. So far it is largely unclear which molecular mechanisms underlie this development.

Effective antiage products show their effect on the broadest possible spectrum of molecular phenomena of skin aging. So far, however, only a few molecular events of skin aging have been described, which can thus serve as a target for cosmetic antiage products. The identification of new aging markers makes it possible to understand the complex processes of intrinsic and extrinsic skin aging and their causal relationships. Only with this knowledge can new concepts for cosmetic antiage products be developed that have an effect on the broad spectrum of extrinsic and intrinsic aging processes in the skin. Each cell type of the skin expresses approximately 15,000 different genes and synthesizes a corresponding number of proteins from it. So far, which genes play a role in skin aging is largely unclear.

The skin consists of several different cell types (fibroblasts, keratinocytes in different differentiation states, melanocytes, Merkel cells, Langerhans cells, hair follicle cells, sweat gland cells etc.), so that the complexity of genes expressed in the skin is very great. It has never been possible to describe this immense complexity. So far, it has not been possible to identify genes from this complexity that are related to skin aging and can serve as molecular markers of skin aging.

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 so far not been available or have been very difficult to access, but they can certainly play a decisive role in aging processes and for the skin's home ostasis.

The entirety of all mRNA molecules that are synthesized by a cell or a tissue at a specific point in time is referred to as a "transcriptome". To date, it has not been possible to describe the complete transcriptome, i.e. the entirety of all transcribed genes, in human skin.

The analysis of gene expression is possible with the quantification of specific mRNA molecules (e.g. Northern blot, RNase protection experiments). However, only a relatively limited number of genes can be measured using these techniques.

The cosmetic antiage products on the market exert their effects on one of the few known markers of extrinsic skin aging, such as collagen synthesis, collagenase activity or collagenase inhibitors.

An understanding of the complex aging processes in the skin and the identification of suitable marker proteins allows the targeted search for substances or combinations of substances with a broad spectrum of anti-aging effects. Product concepts of this kind However, it has not yet been possible to develop it because a large number of skin aging markers are not yet known.

Previous studies on the identification of aging markers were carried out using artificial systems. WO 99/52929 (Lifespan Bioscience Inc.) and the work of Danit, L. et al., (2000), Science 287, pp. 2486-2492 describe the identification of aging markers from isolated and subsequently in vitro cultivated fibroblasts Skin. The cells are no longer in their natural environment in which they divide extremely slowly, but divide relatively quickly and change their gene expression in the process. Another crucial point is the fact that fibroblasts in the skin are exposed to the influence of neighboring skin cells, which is no longer the case with isolated and in vitro cultivated cells.

There is therefore a need to identify as many, preferably all, of the genes that are important for the aging of the skin.

It is therefore an object of the present invention to identify as large a part as possible of the genes which are important for skin aging and / or skin stress. In addition, methods for determining skin stress and / or skin aging are to be provided by means of the identified genes.

This first object is achieved according to the invention by a method (1) for identifying the genes which are important for skin aging and / or skin stress in humans or animals in vitro, characterized in that a) a first mixture of expressed in human or animal skin, ie transcribed and possibly also translated genetically coded factors, ie proteins, mRNA molecules or fragments of proteins or mRNA molecules from young human or animal skin, b) a second mixture of expressed, ie transcribed and optionally in human or animal skin also translates translated genetically coded factors, ie of proteins, mRNA molecules or fragments of proteins or mRNA molecules from old human or animal skin and c) subjecting the mixtures obtained in a) and b) to a serial analysis of gene expression (SAGE), and thereby identifying the genes which are expressed differently (differentially) in old and young skin.

Like the other objects of the invention, the method according to the invention can preferably be applied to human skin, but can also be applied to animal skin and to skin models based on human or animal skin.

The technique of "serial analysis of gene expression" (SAGE ™) was used to record the transcriptome of the skin. This technique simultaneously allows the identification and quantification of all genes expressed in the skin. The comparison of the transcriptome of young skin with the transcriptome of old skin leaves the distinction between relevant and irrelevant genes of skin aging.

Human skin from healthy female donors was used for 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 human skin of different ages were analyzed. The first library comes from a 29-year-old female subject (30048 tags), the other library from a 69-year-old female subject (32840 tags). For further analysis, both SAGE ™ libraries were standardized to the average number of tags (31594 tags). The two libraries were compared to identify genes with age-dependent regulation. As expected for two libraries of the same tissue type, the tag repertoire of the two skin libraries is largely similar.

First of all, it is striking that the library from young skin has a clear overexpression of collagens. Some collagens have several alternative poly adenylations, the resulting tags show consistent results. For example, collagen (I) α1 is overexpressed by a factor of 4-5 in young skin, collagen (I) α2 by a factor of 4, and collagen (III) α 1 by a factor of 8. Other gene classes are those of young skin overexpressed correspond to marker proteins, for example of the cytoskeleton. For example, transgelin, desmin, actin, myosin, calponin and tropomyosin are overrepresented between 6 and 37 times. In contrast, some keratins and other keratinocyte-specific genes are overrepresented in the library from old skin. The epidermal keratins 5, 10 and 14 which are strongly expressed in both libraries are e.g. B. represented about 2 times more strongly in the old skin, while keratin 1, which is also strongly expressed, does not show this tendency. Stratifin, Desmocollin, MMP2 (gelatinase) and CLSP (keratinocyte-specific calmodulin) are 5 to 8 times overrepresented.

Tables 1 to 4 contain a detailed list of the genes determined with the aid of the method according to the invention, which are differentially expressed in old and young skin, stating a serial number in column 1, the tag sequence used in column 2, and the determined relative expression frequency in young skin in column 3, the determined expression frequency in old skin in column 4, the quotient of the frequencies (from column 3 and column 4) in column 5, the significance in column 6, the UniGene accession number in column 7 and a brief description of the Gene or gene product in column 8.

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 young skin than in old skin, 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

The 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 the Internet addresses httD.7 / www.ncbi.nlm.nih.gov / UniGene / Hs.Home.html or http://www.ncbi.nlm.nih.gov/genome/ quide directly accessible. Table 1 lists all genes that are at least 2-fold and less than 5-fold differentially expressed.

Table 2 lists all genes that are at least 5-fold and less than 7-fold differentially expressed.

Table 3 lists all genes that are at least 7-fold and less than 10-fold differentially expressed.

Table 4 lists all genes that are at least 10-fold differentially expressed.

Table 5 lists genes that are at least 2-fold differentially expressed; to which, however, no database entry could be assigned and which can therefore only be identified by the tag sequence in column 2.

Table 6 lists genes that are at least 2-fold differentially expressed and that in column 2 by their UniGene Accession Number, in column 3 by their Swissprot or TREMBL number, or in column 4 by their EMBUGenbank number To be defined.

Table 7 lists genes that are differentially expressed between 2.89 and 11.10-fold. The assignment of the tags to the genes, which are defined by your UniGene Accession Number in column 6, was done by manual annotation.

The following databases were used for the annotation:

1. Unigene version from 10/30/01 with the following database entries: a. the known genes from Genbank (as of October 12, 2001) b. the EST's from dbEST (as of 10/19/01)

2. mRNA version released on 10/17/01

The databases were downloaded from the NCBI, formatted for a local version of the BLAST program (also NCBI) and compared for identical hits with the tags detected in the SAGE analysis. The genes / clones found were checked for redundancy and reworked as follows:

1. Tag sequences with several different hits: Evaluation as not annotable.

2. Tag sequences with double or more identical hits: Elimination of the hits that were furthest away from the poly-A-tail.

First, the results from the Unigene database were evaluated and then compared with the results from the mRNA database. The latter do not appear in Table 7 because they can also be called up via the Unigene entries.

All links listed in the results table were checked on the following documented database of 10/30/2001 (Unigene database release: UniGene Build # 143):

Sequences Included in UniGene

Known genes are from GenBank (Oct 12, 2001) ESTs are from dbEST through 19-Oct-2001

69367 mRNAs + gene CDSs

1147828 EST, 3'reads

1196006 EST, 5'reads

+ 598081 EST, other / unknown

3011282 total sequences in clusters

Final Number of Clusters (sets) 96,332 sets total

20516 sets contain at least one known gene 95 171 sets contain at least one EST 19 355 sets contain both genes and ESTs

Release notes

The genes or gene products defined by their Swissprot or TREMBL numbers are disclosed on the Internet at the following addresses: http://www.ebi.ac.uk/swissprot/ or http://www.ncbi.nlm.nih.gov /.

The genes or gene products defined by their EMBUGenbank numbers are in

Internet revealed at the following address: http://ncbi.nlm.nih.gov/.

The second object underlying the present invention is achieved according to the invention by a method (2) for determining skin stress and / or skin aging in humans or animals, in particular in women, in vitro, which is characterized in that a) a mixture of proteins , mRNA molecules or fragments of proteins or mRNA molecules from human or animal skin, 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 identified as differentially expressed in old and young skin by means of serial analysis of gene expression (SAGE), c) compares the test results from b) with the expression patterns identified by means of serial analysis of gene expression (SAGE) and d) the mixture examined in b) is assigned to old or stressed skin if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed more strongly in old or stressed skin than in young or undressed skin , or assigns the mixture examined in b) to young or undressed skin if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are expressed more strongly in young or undressed skin than in old or stressed skin ,

In step b) of the method for determining skin stress and / or skin aging, 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 by means of serial analysis gene expression (SAGE) can be identified as differentially expressed in old and young skin if these are only expressed in old or only in young skin. 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 skin stress and / or skin aging, the mixture of old or stressed skin examined in b) is assigned if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed more in old or stressed skin than in young or stressed skin, d. that is, the mixture either contains more different compounds typically expressed in old skin than those typically expressed in young skin (qualitative differentiation), or contains more copies of compounds typically expressed in old skin than is typically present in young skin are (quantitative differentiation). The assignment to young or undressed skin is carried out in a complementary manner.

A preferred embodiment of the method according to the invention for determining skin stress and / or skin aging 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 protein NEN or mRNA molecules that are defined in Tables 1 to 4 in column 7 by their UniGene Accession Number, in step c) the test results from b) with those in Tables 1 to 4 in columns 3 and 4 compares the specified expression frequencies and the expression quotients specified in column 5 and in step d) assigns the mixture of old or stressed skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are in old age or stressed skin are expressed at least twice as strongly as in young or undressed skin, or assigns the mixture of young or undressed skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are expressed at least twice as strongly in young or undressed skin as in old or stressed skin.

A further preferred embodiment of the method according to the invention for determining skin stress and / or skin aging 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 Examined mRNA molecules, which are defined in Tables 2 to 4 in column 7 by their UniGene Accession Number, in step c) the test results from b) with the relative given in Tables 2 to 4 in columns 3 and 4 Expression frequencies and the expression quotients indicated in column 5 are compared and in step d) the mixture of old or stressed skin examined in b) is assigned if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are in old or stressed skin are expressed at least 5 times as strongly as in young or undressed skin, or in b) the examined mixture of young or undressed skin is assigned if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed in young or undressed skin at least 5 times as strongly as in old or stressed skin Skin.

Another preferred embodiment of the method according to the invention for determining skin stress and / or skin aging 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 listed in tables 3 and 4 in column 7 by their UniGene accession number in step c) the test results from b) are compared with the relative expression frequencies given in tables 3 and 4 in columns 3 and 4 as well as with the expression quotients given in column 5 and in step d) the mixture of old resp assigned stressed skin if it contains predominantly proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least 7-fold as strongly in old or stressed skin as in young or undressed skin, or that in b ) examined mixture of young or undressed skin assigned if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least 7-fold as strongly in young or undressed skin as in old or stressed skin.

A further preferred embodiment of the method according to the invention for determining skin stress and / or skin aging 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 investigated mRNA molecules, which are defined in Table 4 in column 7 by their UniGene Accession Number, in step c) the test results from b) with the relative expression frequencies given in table 4 in columns 3 and 4 and the column 5 compares the given expression quotient and in step d) assigns the mixture of old or stressed skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are at least 10 times in old or stressed skin are expressed as strongly as in young or undressed skin, or the gemi examined in b) assigned to young or undressed skin if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least 10 times as strongly in young or undressed skin as in old or stressed skin. A further preferred embodiment of the method according to the invention for determining skin stress and / or skin aging 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 investigated, which are defined in Table 5 or in Table 7 in column 2 by their 11-base tag sequence, in step c) the test results from b) with those in Table 5 or in Table 7 in columns 3 and 4 specified relative expression frequencies and the expression quotient indicated in column 5 and in step d) assigns the mixture of old or stressed skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are contained in old or stressed skin at least twice, especially 5 times, preferably 7 times, especially b e are preferably expressed 10 times as strongly as in young or undressed skin, or assigns the mixture of young or undressed skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are in young or undressed skin are expressed at least twice, in particular 5 times, preferably 7 times, particularly preferably 10 times as much as in old or stressed skin.

The condition of the skin can also be described by quantifying several markers (expression products of the genes which are important for skin aging and / or skin stress), which must then be active in a characteristic relationship with one another in order to represent young skin, or in a characteristic ratio different from this must be active in order to represent old skin.

The present invention therefore furthermore relates to a method (3) for determining skin stress and / or skin aging in humans or animals, in particular in women, in vitro, which is characterized in that a) a mixture of proteins, mRNA Molecules or fragments of proteins or mRNA molecules from human or animal skin wins, b) in the mixture obtained at least two of the proteins, mRNA molecules or fragments of proteins or mRNA molecules quantified by means of method (1) as for skin aging and / or the skin stress are significantly identified, c) the expression ratios of the at least two proteins, mRNA molecules or fragments of proteins or mRNA molecules relative to one another are determined, d) the expression ratios from c) are compared with the expression ratios, which are typically young or old for the molecules quantified in b) Skin is present, in particular with the expression ratios that result from Tables 1 to 5, columns 3 and 4, and e) assigns the mixture of old or stressed skin obtained in a) if the expression ratios of the examined skin match the expression ratios in old Correspond to skin, or assign the young or undressed skin mixture obtained in a) if the expression ratios of the examined skin correspond to the expression ratios in young skin.

In step a) of the method according to the invention for determining skin stress and / or skin aging, the mixture is preferably obtained from a skin sample, in particular from a whole skin sample or from an epidermis sample. The whole skin sample opens up more extensive comparison options 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 skin stress and / or skin aging, 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 after the puncture have subsided, the microdialysis delivers 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 whole 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 skin stress and / or skin aging 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

One- or two-dimensional gel electrophoresis Affinity chromatography 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, to which herewith is fully referred to.

2D gel electrophoresis is described, for example, in LD Adams, Two-dimensional Gel Electrophoresis using the Isodalt System or in LD Adams & SR Gallagher, Two-dimensional Gel Electrophoresis using the O'Farrell System; both in Current Protocols in Molecular Biology (1997, Eds. FM Ausubel et al.), Unit 10.3.1-10.4.13; or in 2-D E-lectrophoresis 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 in the art, for example as described in the following references:

Methods in Molecular Biology, 1999; Vol 112; 2-D Proteome Analysis Protocols; Editor: A. J. Link; Humana Press; Totowa; New Jersey. In particular: Courchesne, P.L. and Patterson, S. D .; 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.

A further preferred embodiment of the method according to the invention for determining skin stress and / or skin aging 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 molecules molecular fragments; or in method (3) performs 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), 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. Use of nucleic acid chips, or by means of suitable combinations of these methods.

These methods which can be used according to the invention are described in the overview 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 therein, to which 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.

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.

A further preferred embodiment of the method according to the invention for determining skin stress and / or skin aging 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 which i. in tables 1 to 4 in column 7 by their UniGene Accession Number, or ii. in Table 6 or 8 in column 2 by their UniGene Accession Number, or in column 3 by their Swissprot or TREMBL number, or in column 4 by their EMBL / Genbank number, or in Table 9 by the name of the gene , or the iii. be defined in Table 5 or in Table 7 in column 2 by their 11 base tag sequence.

Another object of the present invention is a test kit for determining skin stress and / or skin aging in humans or animals in vitro, comprising means for carrying out the method according to the invention for determining skin stress and / or skin aging. Another object of the present invention is a biochip for determining skin stress and / or skin aging in humans or animals 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 1 to 4 in column 7 by their UniGene accession number, or which are defined in Table 5 or in Table 7 in column 2 by their 11-base tag sequence.

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 there may be single strands, triplex structures or combinations thereof), saccharides, peptides, proteins (e.g. 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, for example, also be formed 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 (total), 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 previously mentioned 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 therein, to which reference is hereby made in full. The books "DNA Microarrays: A Practical Approach" (editor: Mark Schena, 1999, Oxford University Press) and "Microarray Biochip Technology" (editor: Mark Schena, 2000, Eaton Publishing) provide a clear overview of the practical application methods of DNA chip technology. , 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 for years in Southern blot and Northern blot analysis. 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 production of oligonucleotide arrangements by direct synthesis on a glass surface, which was derivatized with 3-glycidoxypropyltrimethoxysilane and then with a glycol.

A similar method realizes the in situ synthesis of oligonucleotides using a photosensitive, combinatorial chemistry that can be compared with photolithographic techniques (Pease, AC et al. (1994), Proc. Natl Acad Sei USA 91, 5022- 5026). In addition to these techniques based on s / ft / synthesis of oligonucleotides, existing DNA molecules can also be bound to surfaces of support material.

P.O. Brown (DeRisi et al. (1997), Science 278, 680-686) describes the immobilization of DNA on glass surfaces coated with polylysine.

The publication of L.M. Smith (Guo, Z. et al. (1994), Nucleic Acid Research 22, 5456-5465) discloses a similar procedure: oligonucleotides bearing a 5'-terminal amino group can be bound to a glass surface which is coated with 3-aminopropyltrimethoxysilane and then treated with 1,4-phenyldiisothiocyanate.

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 piezo-controlled nanodispenser, which, similar to an inkjet printer, applies probe solutions with a volume of 100 picoliters to the surface of the carrier material in a contact-free manner.

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 primer pair, 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 a typical application for DNA chips, mRNA is isolated from two cell populations to be compared. The isolated mRNAs are converted into cDNA by means of reverse transcription using, for example, fluorescence-labeled nucleotides. The samples to be compared are marked with, for example, red or green fluorescent nucleotides. The cDNAs are then hybridized with the gene probes immobilized on the DNA chip and the bound fluorescence is then quantified.

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 different probes 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.

In a further preferred form, the biochip according to the invention comprises peptide or protein probes, in particular antibodies.

In a further preferred form, the biochip according to the invention comprises 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 listed in Table 6 or 8 in column 2 by their UniGene accession. Number, or in column 3 by their Swissprot or TREMBL number, or in column 4 by their EMBL / Genbank number, or in Table 9 by the name of the gene.

Another object of the present invention is the use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which i. in tables 1 to 4 in column 7 by their UniGene Accession Number, or the ii. in Table 6 or 8 in column 2 by their UniGene Accession Number, or in column 3 by their Swissprot or TREMBL number, or in column 4 by their EMBL / Genbank number, or in Table 9 by the name of the gene , or the iii. in Table 5 or in Table 7 in column 2 are defined by their 11-base tag sequence as stress and / or aging markers of the skin in humans or animals.

Another object of the present invention is a test method for demonstrating the effectiveness of cosmetic or pharmaceutical active substances against skin stress and / or skin aging in vitro, characterized in that a) the skin status is determined by a method according to the invention for determining skin stress and / or skin aging, or using a test kit according to the invention for determining skin stress and / or skin aging, or using a biochip according to the invention, b) applying an active ingredient against skin stress and / or skin aging to the skin one or more times, c) renewing the skin status by means of an inventive method Method for determining skin stress and / or skin aging, or using a test kit according to the invention for determining skin stress and / or skin aging, or using a biochip according to the invention, and d) the effectiveness of the active ingredient by comparing the results from a) and c) determined.

To accelerate the test procedure, it is also possible to apply different active substances or placebos in parallel to different skin areas; for example, an active ingredient on the left forearm and a placebo on the right forearm, or vice versa.

Another object of the present invention is a test kit for demonstrating the effectiveness of cosmetic or pharmaceutical active substances against skin stress and / or skin aging in vitro, 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 i. in tables 1 to 4 in column 7 by their UniGene Accession Number, or ii. in Table 6 or 8 in column 2 by their UniGene Accession Number, or in column 3 by their Swissprot or TREMBL number, or in column 4 by their EMBL / Genbank number, or in Table 9 by the name of the gene , or the iii. in Table 5 or in Table 7 in column 2 are defined by their 11-base tag sequence to demonstrate the effectiveness of cosmetic or pharmaceutical active ingredients against skin stress and / or skin aging.

Another object of the present invention is a screening method for the identification of cosmetic or pharmaceutical active ingredients against skin stress and / or skin aging in vitro, characterized in that a) the skin status by an inventive method for determining the skin stress and / or Skin aging, or by means of a test kit according to the invention for determining skin stress and / or skin aging, or by means of a biochip according to the invention, b) applying a potential active substance against skin stress and / or skin aging to the skin one or more times, c) the skin status again determined by a method according to the invention for determining skin stress and / or skin aging, or by means of a test kit according to the invention for determining skin stress and / or skin aging, or by means of a biochip according to the invention, and d) active ingredients by comparing the results from a ) and c) determined.

Another object of the present invention is the use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which i. in tables 1 to 4 in column 7 by their UniGene Accession Number, or the ii. in Table 6 or 8 in column 2 by their UniGene Accession Number, or in column 3 by their Swissprot or TREMBL number, or in column 4 by their EMBL / Genbank number, or in Table 9 by the name of the gene , or the iii. in Table 5 or in Table 7 in column 2 are defined by their 11-base tag sequence for the identification of cosmetic or pharmaceutical active substances against skin stress and / or skin aging.

Another object of the present invention is a method for producing a cosmetic or pharmaceutical preparation for skin stress and / or skin aging, characterized in that a) active ingredients with the aid of the screening method according to the invention, or the use for the identification of cosmetic or pharmaceutical active ingredients against skin stress and / or skin aging and b) active ingredients found to be effective mixed with cosmetically and pharmacologically suitable and compatible carriers.

The present invention further provides a cosmetic or pharmaceutical preparation for skin stress and / or skin aging, comprising at least one nucleic acid construct which is suitable for suppressing or reducing the activity of at least one of the proteins which are more strongly expressed in old or stressed skin than in young or undressed skin, or to induce or to increase the activity of at least one of the proteins which are expressed more strongly in young or undressed skin than in old or stressed skin.

The proteins are preferably selected from those in Tables 1 to 4 in column 7 by their UniGene accession number, or in Table 6 or 8 in column 2 by their UniGene accession number, or in column 3 their Swissprot or TREMBL number, or in column 4 by their EMBL / Genbank number, or in Table 9 by the name of the gene, or the tag sequence in Table 5 or in Table 7 in column 2 by their 11 bases To be defined. The nucleic acid construct is preferably selected from DNA, RNA or PNA. However, linear combinations of these nucleic acids or hybrid molecules, for example RNA / DNA hybrids, are also possible. The nucleic acid construct is also preferably selected from protein-coding sequences, ribozymes, antisense nucleic acids, triple helix formers and rRNA.

The preparation according to the invention can contain 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 nucleic acid constructs which differ from one another.

The nucleic acid construct is present in the preparation according to the invention in particular enclosed in lipid vesicles, for example in liposomes, niosomes or transfersomes, preferably in liposomes.

Suitable nucleic acid constructs for the purposes of the invention are DNA and RNA sequences which code for one of the age markers and constructs of these polynucleotides.

After being transported into the cells of the skin, these are transcribed and / or translated and lead to an increased expression of the protein encoded by them.

The invention also includes partial sequences of age genes and genes whose sequence has been changed in comparison to the age genes mentioned by directed or other types of mutagenesis.

A number of techniques for modifying cloned genes are known and are described, for example, in "Current Protocols in Molecular Biology", Volume 1, Chapter 6, Ausubel et al. (Ed.), John Wiley & Sons, Ine (2001). These changes can be carried out in such a way that the protein resulting after transcription and / or translation has the identical amino acid sequence as the protein which would have resulted without this mutation (redundancy of the genetic code), but the modified gene can also be used to express a protein with a modified amino acid sequence lead so that its biological activity is changed, for example increased or decreased. In addition, polynucleotides for insertion into the skin are DNA and RNA sequences which comprise part of the sequence of one or more of the genes mentioned and themselves have a desired biological activity (for example anti-sense RNA, rRNA or short double-stranded DNA molecules) ,

Any gene which can be used according to the invention, but preferably those which were found to be weakly expressed in the aging skin, can be introduced into the cells of the skin with the aim of its increased expression.

Any construct from one of the genes which can be used according to the invention, functionally linked to a eukaryotic promoter, can be used.

The constructs with one of the age genes can be any eukaryotic expression constructs. For example, bacterial plasmids, viral constructs, or other DNA constructs can be genetically modified to produce a recombinant DNA (or RNA) molecule that contains a sequence that encodes one of the age genes and expresses the desired gene product in skin cells ,

Constructs which have the ability to replicate in both eukaryotic and prokaryotic host cells are preferred. Such constructs are state of the art and commercially available.

The preparation according to the invention is preferably applied to the skin. The DNA contained in it can be either linear or circular, preferably circular DNA molecules. The polynucleotide or the polynucleotide-containing construct can be reproduced and purified by known methods and is used as a pure molecule or in one of the u.g. Wording used.

Constructs which carry a promoter which enables expression of the DNA of interest are preferred. Depending on the nature of the gene and the purpose of its use, different promoters can be used. Strong, constitutive promoters can be used, such as the immediate early promoter gene "of cytomegalovirus (CMV) or the promoter of the" long terminal repeat "of Rous Sarcoma virus (RSV).

Alternatively, tissue-specific promoters or cell type-specific promoters can be used. For example, the promoter can be selected so that it effects the specific expression in skin cells or certain skin cell types. Examples of tissue-specific or cell-type-specific promoters include the keratin promoters (e.g. human keratin 14 promoter (Wang et al. 1997 Proc. Natl. Acad. Be US 94: 219-226)) or tyrosinase promoters (specific for melanocytes). Alternatively, inducible promoters can be used.

In addition to the promoters, the constructs can also contain other elements which enhance the transcriptional or translational expression of the gene product. For example, the construct may include an "internal ribosomal entry site" (IRES) to enhance translation of the downstream sequence (see Murakami et al. 1997, Gene 202: 23-29). Other components that are present on the construct may include markers (e.g., antibiotic resistance genes such as the ampicillin resistance gene) for selecting cells containing the construct, an origin of replication that causes the construct to replicate stably in prokaryotic cells, a nuclear localization signal, or other elements that inhibit the production of the construct Support DNA construct and / or the encoded protein.

The constructs can also contain a polyadenylation signal. The sequence of such a signal can be selected from various known polyadenylation signals. A preferred example is the SV40 early polyadenylation signal.

The constructs can also include one or more introns that can enhance expression of the DNA.

The invention also includes constructs that code for fusion proteins of one of the age markers with a second protein or for a fusion protein of two age markers. Preferred nucleic acid constructs are those with a plurality of expression cassettes, on which two or more age markers are coded, or which code one of the age genes and a gene for a further protein.

Co-transfection with one or more further vectors which can support the expression of the gene of interest is also possible.

Vectors which carry one or more of the features mentioned and, in addition, can also contain further functional units have been described many times in the literature and are commercially available. Such vectors are e.g. pCI and pSI (Promega GmbH) or pDEST (Gibco BRL).

The sequences of the age genes and partial sequences of the genes which can be determined according to the invention and in particular those listed in Tables 1 to 9 can be used for the selective inhibition of the expression of individual genes.

Preferred targets for the inhibition of expression are those which have been found to be expressed more strongly in the aging skin. Oligonucleotides and polynucleotides suitable for this purpose include antisense nucleotides, ribozyme nucleotides and double-stranded RNAs.

Antisense nucleotides are well known in their ability to hybridize with sense strands of the mRNA and thereby interfere with the expression of the mRNA (see, for example, Wingers et al., Laboratory Investigation 79, 1415-1424 (1999). An overview of the use of antisense nucleotides in the skin gives Wraight et al., Pharmacol & Ther 90, 89-104 (2001).

Ribozyme nucleic acids are also known as single-stranded RNA molecules that have the ability to selectively cleave ssRNA and ssDNA, and thereby selectively inhibit the expression of the target molecules.

Post-transcriptional gene repression can also be caused by double-stranded RNAs. These dsRNAs will soon interfere with the target RNA after cleavage. right segments by ribonuclease III. Duplexes from short RNA sequences can also be used for gene repression (SM Elbashir et al., Nature 411, 494-498 (2001). These double-stranded RNA molecules can also be used in the sense of the invention for the repression of the found age genes.

Unmodified DNA or RNA molecules are rapidly degraded in tissues or cells. In order to increase the resistance of oligo- or polynucleotides to degradation by nucleases, modifications have been developed which affect either the backbone or the pyridine or pyrimidine bases of an oligonucleotide. Such modified DNA or RNA sequences can also be used for the purposes of the invention. Suitable modifications are e.g. Phosphorothioates, methylphosphonates or peptide linkages (PNAs) for the sugar backbone as well as C5-propynyl-dU, dC for the nucleoside bases.

The DNA or RNA molecules of the invention can e.g. can be applied topically. The molecules can either be used without further substances influencing the penetration, or in a mixture or associated with molecules which influence the penetration through the stratum corneum and the transfection of the cells of the skin.

A preferred embodiment of the topical application of the age genes is that in a formulation with lipids, optionally in a mixture with surfactants, preferably in the form of liposomes.

The formulation contains about 0.1 μg - 5 mg DNA or RNA per mg liposome. The components of the liposomes can be neutral or charged and in the form of e.g. multilamellar vesicles or unilamelar vesicles are present.

Suitable lipids for the production of liposomes are, for example, natural phosphatidylcholine, which can be obtained, for example, from egg, soybean, olives, coconut, whale fat, saffron, linseed, evening primrose or primrose. Other suitable lipids are, for example, natural or synthetic phosphatidylethanolamine, synthetic phosphatidylcholine, phosphatidic acids or their esters, phosphatidylserine and phosphatid dyl (poly) alcohols, such as phosphatidylinisitol or phosphatidylglycerol. Examples of the lipids mentioned are DPPC (dipalmitoylphosphatidylcholine), DOPE (dioleylphosphatidylethanolamine), DOTMA (N [1- (2,3, dioleoyloxy) propyl] -N, N, N trimethylammonium chloride), DOTAB (N-1- (2,3-Dioleoyloxy) propyl N, N, N-trimethylammonium chloride), DPPA (dipalmitoylphosphatidic acid), DPPG (dipalmitoylphosphatidylglycerol).

Individual surface-active compounds, which are used, for example, as detergents or emulsifiers, can also be used to form liposomes. Examples include DODAC (dioctadecylammonium chloride) and CTAC (cetyltrimethylammonium chloride). Suitable constituents of liposomes and production processes are described in the literature (see, for example, “Liposome Drug Delivery Systems”, G. Betageri (ed.), Lancaster Techonomic Publishing Company (1993) or “Liposome Technology”, Gregoriadis (ed.), CRC Press ). In addition to liposomes, other lipid vesicles such as niosomes and transfersomes (see e.g. WO9817255) can also be used.

The penetration ability of the DNA and RNA molecules can be improved by the previous or simultaneous application of penetration enhancers. Chemical penetration enhancers have been extensively described in the literature (see, e.g., M. Foldvari, PSTT 3, 417-425 (2000) or N. Kanikkannan, Curr Med. Chem. 7, 593-608 (2000)). Suitable penetration enhancers include organic solvents (eg ethanol), pyrrolidones, sulfoxides (eg DMSO), fatty acids (saturated or unsaturated, branched or unbranched with a preferred chain length of 8-18), terpenes (eg L-menthol or 1,8-cineol) surfactants (e.g. polysorbates (Tween), polyethylene alkylphenols (Brij), alkyl ether sulfates as well as betaines and amphoteric glycinates).

Furthermore, invasive or minimally invasive methods are also possible for improving the penetration of oligo- or polynucleotides in the sense of the invention. Common minimally invasive methods include electroporation and iontophoresis. In both methods, a voltage is applied to the surface of the skin with the help of electrodes. Electroporation uses a short pulse of high voltage to permeate the skin. For this purpose, a small voltage with constant current is used in iontophoresis. Low frequency ultrasound can also be used to increase skin permeability to DNA or RNA.

The following exemplary embodiments illustrate the invention, but without restricting it thereto:

example 1

Preparation of liposome-DNA complexes

18.6 μl (25 μmol) DOPE and 0.0175 g DOTAP (25 μmol) are dissolved together in 1 ml ethanol. The solution is dried in an eksikkator overnight and the lipid mixture is then rehydrated in 3 ml PBS (58 mM NaHPO ₄ , 17 mM NaH ₂ PO ₄ , 69 mM NaCl, pH 7.4) with exclusion of light for 3-4 h with occasional mixing. The resulting lipid dispersion is sonicated twice for 5 min each in an ultrasonic bath with an intermittent 2-hour break. 6.1 μl of the resulting liposome dispersion are diluted with 1.5 ml PBS and mixed with 20 μg plasmid dissolved in 1.5 ml HBS (150 mM NaCl, 20 mM Hepes, pH 7.4). The greater turbidity which results when the plasmid solution is added to the liposome dispersion gives an indication of the formation of the DNA-liposome complexes.

Example 2

Preparation of liposome-DNA complexes

18.6 μl (25 μmol) DOPE and 0.0175 g DOTAP (25 μmol) are dissolved together in 1 ml ethanol. The solution is dried in an eksikkator overnight and the lipid mixture is then rehydrated in 3 ml PBS (58 mM NaHPO ₄ , 17 mM NaH ₂ PO ₄ , 69 mM NaCl, pH 7.4) with exclusion of light for 3-4 h with occasional mixing. The resulting lipid dispersion is sonicated twice for 5 min each in an ultrasonic bath with an intermittent 2-hour break. 50 μl of the resulting liposome dispersion are mixed with 50 μg plasmid dissolved in 50 μl HBS (150 mM NaCl, 20 mM Hepes, pH 7.4). The liposomes are detected by TEM images of the liposome-DNA complexes (FIG. 1) - Example 3

Production of liposomes with cationic detergent and complex formation with DNA

29.8 μl (40 μmol) DOPE and 4 mg CTAC (cetyltrimethylammonium chloride) (10 μmol) are dissolved together in 1 ml ethanol. The solvent ethanol is removed in a rotary evaporator and the mixture is then rehydrated in 3 ml PBS (58 mM NaHPO ₄ , 17 mM NaH ₂ PO ₄ , 69 mM NaCl, pH 7.4) with exclusion of light for 3-4 h with occasional mixing. The resulting lipid dispersion is sonicated twice for 5 min each in an ultrasonic bath with an intermittent 2-hour break. 6.7 μl of the resulting liposome dispersion are diluted with 1.5 ml PBS and mixed with 20 μg plasmid dissolved in 1.5 ml HBS (150 mM NaCl, 20 mM Hepes, pH 7.4).

List of figures:

Figure 1: TEM image of the liposome-DNA complexes from Example 2 in cryomode. Magnification: 10000 times

Figure 1:

tables:

Table 4:

Table 3:

Table 2:

Table 1:

Table 5:

Table 7:

Table 6:

Table 8:

TRANSPORTER 1. (4F2 LC) 4F2 LIGHT CHAIN. (SLC7A5).

144 Hs.73965 Q01130 MRF1: (SFRS2) SPLICING FACTOR, ARGININE / SERINE- RICH 2 (SPLICING FACTOR SC35) (SC-35) (SPLICING COMPONENT, 35 KDA) (PR264 PROTEIN).

145 Hs. 25313 014742 MSP58: (MSP58) NUCLEOLAR PROTEIN CELL CYCLE-REGULATED FACTOR P78.

146 Hs.76941 P54709 NA-K-ATPASE-B3: (ATP1 B3) SODIUM / POTASSIUM-TRANSPORTING ATPASE BETA-3 CHAIN (SODIUM / POTASSIUM-DEPENDENT ATPASE BETA-3 SUBUNIT) (ATPB-3).

147 ms. 15977 Q9Y6M9 NDUFB9: (NDUFB9 OR UQOR22) NADH-UBIQUINONE OXIDOREDUCTASE B22 SUBUNIT (EC 1.6.5.3) (EC 1.6.99.3) (COMPLEX 1-B22) (CI-B22).

148 ms.172674 Q99842 NFATX4: (NFATC3 OR NFAT4) NUCLEAR FACTOR OF ACTIVATED T-CELLS, CYTOPLASMIC 3 (T CELL TRANSCRIPTION FACTOR NFAT4) (NF-ATC3) (NF-AT4) (NFATX).

149 Hs.62041 P14543 NIDOGEN: (NID) NIDOGEN PRECURSOR (ENTACTIN).

150 Hs.111039 P30419 NMT1: (NMT1 OR NMT) GLYCYLPEPTIDE N- TETRADECANOYLTRANSFERASE 1 (EC 2.3.1.97) (PEPTIDE N-MYRISTOYLTRANSFERASE 1) (MYRISTOYL-COA.PROTEIN N- MYFERISTASE) (NM

151 Hs.724 P20393 NR1D1: (NR1D1 OR THRAL OR EAR1 OR HREV) ORPHAN NUCLEAR RECEPTOR NR1D1 (V-ERBA RELATED PROTEIN EAR-1) (REV-ERBA-ALPHA).

152 Hs.1119 P22736 NR4A1: (NR4A1 OR HMR OR NAK1 OR GFRP1) ORPHAN NUCLEAR RECEPTOR HMR (EARLY RESPONSE PROTEIN NAK1) (TR3 ORPHAN RECEPTOR)

153 Hs.82120 P43354 NR4A2: (NR4A2 OR NURR1 OR TINUR OR NOT) ORPHAN NUCLEAR RECEPTOR NURR1

154 Hs.83469 Q14494 NRF1: (NFE2L1 OR NRF1 OR TCF11 OR HBZ17) NUCLEAR FACTOR ERYTHROID 2 RELATED FACTOR 1 (NF-E2 RELATED FACTOR 1) (NFE2-RELATED FACTOR 1) (NUCLEAR FACTOR, ERYTHROID DERIVED 2, LIKE TRANSCRIPTION FACTOR 11) (TRANSCRIPTION FACTOR HBZ17)

155 Hs.75212 P11926 ODC1: (ODC1) ORNITHINE DECARBOXYLASE (EC 4.1.1.17) (ODC).

156 Hs. 88474 P23219 PGH1: (PTGS1 OR COX1) PROSTAGLANDIN G / H SYNTHASE 1 PRECURSOR (EC 1.14.99.1) (CYCLOOXYGENASE-1) (COX-1) (PROSTAGLANDIN- ENDOPEROXIDE SYNTHASE 1) (PROSTAGNESSINE H2SYNTHASE 1) 1) (PGHS-1) (PHS 1).

157 Hs.76152 P07585 PGS2: (DCN) BONE PROTEOGLYCAN II PRECURSOR (PG-S2) (DECORIN) (PG40).

158 Hs. 9589 Q9UMX0 PLIC-1: (UBQLN1) PLIC-1 UBIQUILIN. (DA41)

159 Hs.173902 P30153 PPP2R1A: (PPP2R1A) SERINE / THREONINE PROTEIN PHOSPHATASE 2A, 65 KDA REGULATORY SUBUNIT A, ALPHA ISOFORM (PP2A, SUBUNIT A, PR65-ALPHA ISOFORM) (PP2A, SUBUNIT A, R1-ALPHA TEMPERAMENT) ANTIGEN-ASSOCIATED 61 KDA PROTEIN)

160 P34062 PSMA6: (PSMA6 OR PROS27) PROTEASOME IOTA CHAIN (EC 3.4.99.46) (MACROPAIN IOTA CHAIN) (MULTICATALYTIC ENDOPEPTIDASE COMPLEX IOTA CHAIN) (27 KDA PROSOMAL PROTEIN) (PROS-27) (P27K).

Table 9:

Claims

claims:

1. A method for identifying the genes of importance for skin aging and / or skin stress in humans or animals in vitro, characterized in that a) a first mixture of expressed in human or animal skin, d. H. transcribed and optionally also translated genetically coded factors, that is proteins, mRNA molecules or fragments of proteins or mRNA molecules from young human or animal skin, b) a second mixture of expressed in human or animal skin, d. H. transcribed and possibly also translated genetically coded factors, i.e. proteins, mRNA molecules or fragments of proteins or mRNA molecules from old human or animal skin, and c) the mixtures obtained in a) and b) of a serial analysis of gene expression (SAGE ) and thereby identifies the genes that are expressed differently (differentially) in old and young skin.

2. A method for determining skin stress and / or skin aging in humans or animals in vitro, characterized in that a) a mixture of proteins, mRNA molecules or fragments of proteins or mRNA molecules is obtained from human or animal skin, 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 identified by means of serial analysis of gene expression (SAGE) as being differentially expressed in old and young skin, c) comparing the test results from b) with the expression patterns identified by means of serial analysis of gene expression (SAGE) and d) assigning the mixture of old or stressed skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or contains mRNA molecules that are more strongly expressed in old or stressed skin than in young ones r or undressed skin, or the mixture of young or undressed skin examined in b) if it predominantly contains proteins, mRNA Contains molecules or fragments of proteins or mRNA molecules that are expressed more strongly in young or undressed skin than in old or stressed skin.

3. The method according to claim 2, 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 listed in Tables 1 to 4 in column 7 are defined by their UniGene accession number, in step c) compares the test results from b) with the relative expression frequencies given in tables 1 to 4 in columns 3 and 4 and the expression quotients given in column 5 and in step d) assigns the mixture of old or stressed skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least twice as strongly in old or stressed skin as in young or undressed skin, or assigns the mixture of young or undressed skin examined in b) if it contains predominantly proteins, mRNA moles contains eccles or fragments of proteins or mRNA molecules which are expressed at least twice as strongly in young or undressed skin as in old or stressed skin.

4. The method according to claim 2 or 3, characterized in that 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 which are examined in the Tables 2 to 4 in

Column 7 are defined by their UniGene Accession Number, in step c) the test results from b) with the relative expression frequencies given in tables 2 to 4 in columns 3 and 4 as well as in

Column 5 compares the expression quotient and assigns in step d) the mixture of old or stressed skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are in old or stressed skin are expressed at least 5 times as strongly as in young or undressed skin, or in b) investigated mixture of young or undressed skin is assigned if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least 5 times as strongly in young or undressed skin as in old or stressed skin.

5. The method according to any one of claims 2 to 4, 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 in tables 3 and 4 in column 7 are defined by their UniGene Accession Number, in step c) the test results from b) with the relative expression frequencies given in tables 3 and 4 in columns 3 and 4 as well as in column 5 compares the stated expression quotient and in step d) assigns the mixture of old or stressed skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which have at least 7 in old or stressed skin -expressed as strongly as in young or undressed skin, or assigns the mixture of young or undressed skin examined in b) if it is predominantly P contains roteine, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least 7-fold as strongly in young or undressed skin as in old or stressed skin.

6. The method according to any one of claims 2 to 5, 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, the defined in Table 4 in column 7 by their UniGene Accession Number, in step c) comparing the test results from b) with the relative expression frequencies given in table 4 in columns 3 and 4 and the expression quotients given in column 5 and in step d) the mixture examined in b) is assigned to old or stressed skin if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are at least 10-fold in old or stressed skin are expressed as strongly as in young or undressed skin, or the mixture of young or undressed skin examined in b) is assigned if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are in young or undressed Skin are expressed at least 10 times as strongly as in old or stressed skin.

7. The method according to claim 2, 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 in Table 5 or 7 are defined in column 2 by their 11-base tag sequence, in step c) the test results from b) are compared with the relative expression frequencies given in columns 5 and 7 in columns 3 and 4 and the expression quotients given in column 5 and in Step d) assigns the mixture of old or stressed skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules, which in old or stressed skin at least twice, in particular 5 times , preferably 7-fold, particularly preferably 10-fold, very particularly preferably 11-fold, expressed as strongly as in young or undressed skin, or the i nb) the examined mixture of young or undressed skin is assigned if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are at least twice, in particular 5 times, preferably 7 times, especially in young or undressed skin preferably expressed 10 times as strongly as in old or stressed skin.

8. A method for determining skin stress and / or skin aging in humans or animals in vitro, characterized in that a) a mixture of proteins, mRNA molecules or fragments of proteins or mRNA molecules is obtained from human or animal 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 as being important for skin aging and / or skin stress 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 relative to one another are determined, d) the expression ratios from c) are compared with the expression ratios, which are typically young or old for the molecules quantified in b) Skin is present, in particular with the expression ratios that result from Tables 1 to 5 and 7, columns 3 and 4, and e) assigns the mixture of old or stressed skin obtained in a) if the expression ratios of the examined skin correspond to the expression ratios correspond in old skin, or assign the young or undressed skin mixture obtained in a) if the expression ratios of the examined skin correspond to the expression ratios in young skin.

9. The method according to any one of claims 1 to 8, characterized in that in step a) the mixture from a skin sample, in particular from a whole skin sample or from an epidermis sample is obtained.

10. The method according to any one of claims 2 to 8, characterized in that in step a) the mixture is obtained by means of microdialysis.

11. The method according to any one of claims 2 to 7, 9 and 10, characterized in that one carries out the investigation in step b) for the presence and optionally the amount of at least one of the 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.

12. The method according to any one of claims 8 to 10, characterized in that one carries out the investigation in step b) the quantification of at least two proteins or protein fragments by means of a method which is selected from

13. The method according to any one of claims 2 to 7, 9 and 10, 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 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) and especially xii. Use of nucleic acid chips, or by means of suitable combinations of these methods.

14. The method according to any one of claims 8 to 10, 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),

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. Use of nucleic acid chips, or by means of suitable combinations of these methods.

15. The method according to any one of claims 2 to 7, 9, 10, 11 and 13, 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 up 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 i. in tables 1 to 4 in column 7 by their UniGene Accession Number, or ii. in Table 6 or 8 in column 2 by their UniGene Accession Number, or in column 3 by their Swissprot or TREMBL number, or in column 4 by their EMBL / Genbank number, or in Table 9 by the name of the gene , or the iii. be defined in Table 5 or in Table 7 in column 2 by their 11 base tag sequence.

16. The method according to any one of claims 8 to 10, 12 and 14, characterized in that in step b) 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 up 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 i. in tables 1 to 4 in column 7 by their UniGene Accession Number, or ii. in Table 6 or 8 in column 2 by their UniGene Accession Number, or in column 3 by their Swissprot or TREMBL number, or in column 4 by their EMBL / Genbank number, or in Table 9 by the name of the gene , or the iii. be defined in Table 5 or in Table 7 in column 2 by their 11 base tag sequence.

17. Test kit for determining skin stress and / or skin aging in humans or animals in vitro, comprising means for carrying out the method according to one of claims 2 to 16.

18. Biochip for determining skin stress and / or skin aging in humans or animals in vitro, comprising i. a carrier 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 1 to 4 in column 7 by their UniGene accession number, or which are defined in Table 5 or in Table 7 in column 2 by their 11-base tag sequence.

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. 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. Biochip according to one of claims 18 to 22, comprising 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 in Table 6 or 8 in column 2 by their UniGene -Accession number, or in column 3 by their Swissprot or TREMBL number, or in column 4 by their EMBL / Genbank number, or in table 9 by the name of the gene,

24. Use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which i. in tables 1 to 4 in column 7 by their UniGene Accession Number, or ii. in Table 6 or 8 in column 2 by their UniGene Accession Number, or in column 3 by their Swissprot or TREMBL number, or in column 4 by their EMBL / Genbank number, or in Table 9 by the name of the gene , or the iii. in Table 5 or in Table 7 in column 2 are defined by their 11-base tag sequence as stress and / or aging markers of the skin in humans or animals.

25. Test method for proving the effectiveness of cosmetic or pharmaceutical active substances against skin stress and / or skin aging in vitro, characterized in that a) the skin status by a method according to one of claims 2 to 16, or by means of a test kit according to claim 17 , or determined by means of a biochip according to one of claims 18 to 23, b) applying an active substance against skin stress and / or skin aging to the skin one or more times, c) again the skin status by a method according to one of claims 2 to 16, or by means of a test kit according to claim 17, or by means of a biochip according to of claims 18 to 23, and d) determines the effectiveness of the active ingredient by comparing the results from a) and c).

26. Test kit for demonstrating the effectiveness of cosmetic or pharmaceutical active substances against skin stress and / or skin aging in vitro, comprising means for carrying out the method according to claim 25.

27. Use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which i. in tables 1 to 4 in column 7 by their UniGene Accession Number, or ii. in Table 6 or 8 in column 2 by their UniGene Accession Number, or in column 3 by their Swissprot or TREMBL number, or in column 4 by their EMBL / Genbank number, or in Table 9 by the name of the gene , or the iii. in Table 5 or in Table 7 in column 2 are defined by their 11-base tag sequence to demonstrate the effectiveness of cosmetic or pharmaceutical active ingredients against skin stress and / or skin aging.

28. Screening method for identifying cosmetic or pharmaceutical active substances against skin stress and / or skin aging in vitro, characterized in that a) the skin status by a method according to one of claims 2 to 16, or by means of a test kit according to claim 17 , or determined by means of a biochip according to one of Claims 18 to 23, b) applying a potential active substance against skin stress and / or skin aging to the skin one or more times, c) the skin status is determined again by a method according to one of claims 2 to 16, or by means of a test kit according to claim 17, or by means of a biochip according to one of claims 18 to 23, and d) active ingredients are compared by comparing the results a) and c) determined.

29. Use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules that i. in tables 1 to 4 in column 7 by their UniGene Accession Number, or ii. in Table 6 or 8 in column 2 by their UniGene Accession Number, or in column 3 by their Swissprot or TREMBL number, or in column 4 by their EMBL / Genbank number, or in Table 9 by the name of the gene , or the iii. in Table 5 or in Table 7 in column 2 are defined by their 11-base tag sequence for the identification of cosmetic or pharmaceutical active substances against skin stress and / or skin aging.

30. A process for the preparation of a cosmetic or pharmaceutical preparation for skin stress and / or skin aging, characterized in that a) active ingredients are determined using the method according to claim 28, or the use according to claim 29, and b) active ingredients found to be effective with cosmetic and pharmacologically suitable and compatible carriers are mixed.

31. Cosmetic or pharmaceutical preparation for skin stress and / or skin aging, comprising at least one nucleic acid construct which is suitable for suppressing or reducing the activity of at least one of the proteins which are expressed more in old or stressed skin than in young or undressed Skin, or to induce or enhance the activity of at least one of the proteins that are more strongly expressed in young or undressed skin than in old or stressed skin.

32. Preparation according to claim 31, characterized in that the protein is selected or the proteins are selected from those which a) in Tables 1 to 4 in column 7 by their UniGene Accession Number, or b) in Table 6 or 8 in column 2 by their UniGene Accession Number, or in column 3 by their Swissprot or TREMBL number, or in column 4 by their EMBL / Genbank number, or in Table 9 by the name of the gene, or c) defined in Table 5 or in Table 7 in column 2 by their 11-base tag sequence.

33. Preparation according to claim 31 or 32, characterized in that the nucleic acid construct is selected from DNA, RNA or PNA.

34. Preparation according to one of claims 31 to 33, characterized in that they are 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 from each other contains various nucleic acid constructs.

35. Preparation according to one of claims 31 to 34, characterized in that the nucleic acid construct is selected from protein-coding sequences, ribozymes, antisense nucleic acids, triple helix formers and rRNA.

36. Preparation according to one of claims 31 to 35, characterized in that the nucleic acid construct is enclosed in lipid vesicles, for example in liposomes, niosomes or transfersomes, preferably in liposomes.