WO2004059001A2 - Method for determining markers of human facial skin - Google Patents
Method for determining markers of human facial skin Download PDFInfo
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- WO2004059001A2 WO2004059001A2 PCT/EP2003/014068 EP0314068W WO2004059001A2 WO 2004059001 A2 WO2004059001 A2 WO 2004059001A2 EP 0314068 W EP0314068 W EP 0314068W WO 2004059001 A2 WO2004059001 A2 WO 2004059001A2
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- skin
- proteins
- mrna molecules
- facial skin
- fragments
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6881—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from skin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/148—Screening for cosmetic compounds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Definitions
- the present invention relates to a method for determining markers of human facial skin in vitro, test kits and biochips for determining markers of human facial skin and the use of proteins, mRNA molecules or fragments of proteins or mRNA molecules as markers of human facial skin; furthermore a test method for the detection of the effectiveness of cosmetic or pharmaceutical active substances for the treatment of human facial skin as well as a screening method for the identification of cosmetic or pharmaceutical active substances for the treatment of human facial skin and a method for the production of a cosmetic or pharmaceutical preparation for the treatment of human facial 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.
- the human genome contains between 30,000 and 140,000 genes.
- 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.
- 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.
- 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. It has so far not been possible to identify from this complexity the genes which are associated with the facial skin and which can serve as molecular markers of this tissue. 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 for analysis techniques or have been very difficult to access, but they can certainly play a decisive role in the facial skin.
- 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
- the transcriptome i.e. the entirety of all transcribed genes of human facial skin, has not been described to date.
- Transcriptome analyzes of the skin using various methods, including SAGE TM analysis, are state of the art. However, isolated keratinocytes (in vitro) or epidermal explants are used, which - as explained above - do not represent models representative of the complex process in the skin.
- DE-A-101 00 127.4-41 From DE-A-101 00 127.4-41 by the applicant it is known to subject skin cells to a SAGE TM 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 TM analysis between stressed or aged skin and undressed or young skin.
- information on specific markers of human facial 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.
- the object of the present invention is therefore to identify as large as possible a part of the genes which are important for the human facial skin.
- the identified genes are also intended to provide methods for determining the homeostasis of human facial skin.
- This first object is achieved according to the invention by a method (1) for identifying the genes which are important for human facial skin in humans in vitro, characterized in that a) a first mixture of genetically coded, ie transcribed and optionally also translated, expressed in human facial skin Factors, ie proteins, mRNA molecules or fragments of proteins or mRNA molecules from human facial skin, b) a second mixture of areas expressed in other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin, ie expressed transcribed and possibly also translated genetically coded factors, that is, proteins, mRNA molecules or fragments of proteins or mRNA molecules from other human tissues (not facial skin), in particular from skin-protected areas, preferably from breast 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 human facial skin.
- SAGE serial analysis of gene expression
- the method according to the invention advantageously makes it possible to understand the complex process of cellular reactions to the environment and the underlying causal relationships in the facial skin. With this knowledge, new concepts for cosmetic facial products can be developed that have an effect on the broad spectrum of gene expression in facial skin.
- the SAGE TM analysis carried out as part of the method according to the invention shows for the first time which genes are expressed in facial skin and which genes are expressed there differently than in other tissues, in particular in the skin of protected areas, such as e.g. the breast skin.
- the entirety of all m-RNA molecules that are synthesized by a cell or a tissue at a specific point in time is referred to as a "transcriptome”.
- the technique of "serial analysis of gene expression” was used to record the transcriptome of the facial skin. This technique simultaneously allows the identification and quantification of all genes expressed in the facial skin.
- the analysis of the gene expression is also possible with the quantification of specific mRNA Molecules are possible (eg Northem blot, RNase protection experiments), but these techniques can only measure a relatively limited number of genes Techniques MPSS (Massive Parallel Signiture Sequencing) or techniques based on differential display that replace SAGE TM analysis. In practice, however, the SAGE TM technology has so far been faster and more reliable than alternative methods and is therefore preferable.
- transcriptome of human facial skin with the transcriptome of other human tissues (not facial skin), in particular the skin of protected areas, preferably the breast skin, allows the distinction between genes relevant and irrelevant to the facial skin. These can be genes that are particularly strongly expressed in facial skin or genes that are characterized in that they are only expressed slightly compared to the breast skin.
- the proportion of mRNA species that are present in a maximum of 5 copies per cell is around 87%.
- This proportion of low-expressed genes suggests that the expression of facial skin-specific genes could also be represented by around 87% by low-expressed genes.
- SAGE TM analyzes In order to record facial skin-specific gene expression, even small, statistically insignificant differences can be included in the evaluation by means of further relevant SAGE TM analyzes.
- gene expression data determined on the basis of SAGE TM analyzes can be compared with a publicly accessible SAGE TM database (CGAP), which consists of approx. 2.5 million tags, all of which do not come from the skin.
- CGAP database can be viewed online at the NCBI at the URL http://www.ncbi.nlm.nih.gov/SAGE.
- the present application has the CGAP database in a version dated
- the database contains 66 projects that were obtained from the URL ftp://ftp.ncbi.nlm.nih.gov/pub/sage/fasta.
- both SAGE TM libraries were normalized to the average number of tags, compared with one another and genes with a facial skin-specific regulation were identified.
- the tag repertoire of the two skin libraries is largely similar.
- Tables 1 to 4 contain a detailed list of the genes which are determined with the aid of the method according to the invention and which are differentially expressed in facial skin and in other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin • a serial number in column 1,
- Tables 5 to 12 contain a detailed list of the genes determined with the aid of the method according to the invention and differentially expressed in facial skin and in other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin
- the quotient in column 3 indicates the strength of the differential expression, ie the factor by which the respective gene is expressed more strongly in facial skin (face) than in breast skin (breast), or vice versa.
- the quotient in column 5 indicates the strength of the differential expression, ie by which factor the respective gene is expressed more strongly in facial skin (face) than in other human tissues (except skin) whose expression profiles are represented by the CGAP database, or the other way around.
- Table 1 lists all genes that are at least 10-fold differentially expressed in facial skin (face) compared to breast skin (breast) with a p-value of p> 0.05 (signif> 1.3).
- Table 2 lists all genes that are expressed differentially in facial skin (face) compared to breast skin (breast) with a p-value of p> 0.05 (Signif> 1, 3) at least 5-fold.
- Table 3 lists all genes that are at least 3 times differentially expressed in facial skin (face) compared to breast skin (breast) with a p-value of p> 0.05 (signif> 1.3).
- Table 4 lists all genes that are at least 1.9 times differentially expressed in facial skin (face) compared to breast skin (breast) with a p-value of p> 0.05 (signif> 1.3).
- Table 5 lists all genes that are at least 5 times differentially expressed in facial skin (Face) compared to breast skin (Breast) with a p-value of p ⁇ 0.05 (Signif ⁇ 1, 3) and those in facial skin (Face) compared to other human tissues (except skin), whose expression profiles by the CGAP database are represented with a p-value of p> 0.05 (Signif> 1.3) are expressed at least 5-fold differentially.
- the comparison of the subsignificant face / breast data with independent SAGE TM data (face / CGAP) confirms the differential gene expression and validates the markers of the facial skin.
- Table 6 lists all genes that are expressed differentially in facial skin (face) compared to breast skin (breast) with a p-value of p ⁇ 0.05 (Signif ⁇ 1, 3) and that in facial skin (Face) compared to other human tissues (except skin), whose expression profiles are represented by the CGAP database, with a p-value of p ⁇ 0.05 (Signif ⁇ 1, 3) are expressed at least 5-fold differentially, whose expression differs by less than a power of ten, i.e. the quotient (Face / Breast) / (Face / CGAP) is less than 10 or greater than 0.1.
- the comparison of the subsignificant face / breast data with independent SAGE TM data (face / CGAP) confirms the differential gene expression and validates the markers of the facial skin.
- Table 7 lists all genes that are at least 3 times differentially expressed in facial skin (Face) compared to breast skin (Breast) with a p-value of p ⁇ 0.05 (Signif ⁇ 1, 3) and those in facial skin (Face) compared to other human tissues (except skin), whose expression profiles are represented by the CGAP database, with a p-value of p> 0.05 (Signif> 1, 3) are expressed at least 3-fold differentially.
- the comparison of the subsignificant face / breast data with independent SAGE TM data (face / CGAP) confirms the differential gene expression and validates the markers of the facial skin.
- Table 8 lists all genes that are at least 3 times differentially expressed in facial skin (Face) compared to breast skin (Breast) with a p-value of p ⁇ 0.05 (Signif ⁇ 1, 3) and that in facial skin (Face) compared to other human tissues (except skin), whose expression profiles are represented by the CGAP database, with a p-value of p ⁇ 0.05 (signif ⁇ 1.3) are expressed differentially at least three times, the expression of which differs by less than a power of ten, i.e. the quotient (face / breast) / (face / CGAP) is less than 10 or greater than 0.1.
- the comparison of the subsignificant face / breast data with independent SAGE TM data (face / CGAP) confirms the differential gene expression and validates the markers of the facial skin.
- Table 9 lists all genes which are expressed differentially in facial skin (Face) compared to breast skin (Breast) with a p-value of p ⁇ 0.05 (Signif ⁇ 1.3) and which in facial skin (face) compared to other human tissues (except skin), whose expression profiles are represented by the CGAP database, with a p-value of p> 0.05 (Signif> 1.3) at least 1.9 times be differentially expressed.
- the comparison of the subsignificant face / breast data with independent SAGE TM data (face / CGAP) confirms the differential gene expression and validates the markers of the facial skin.
- Table 10 lists all genes that are expressed differentially in facial skin (Face) compared to breast skin (Breast) with a p-value of p ⁇ 0.05 (Signif ⁇ 1.3) and that in facial skin (face) compared to other human tissues (except skin), whose expression profiles are represented by the CGAP database, with a p-value of p ⁇ 0.05 (Signif ⁇ 1, 3) at least 1.9 times are differentially expressed, the expression of which differs by less than a power of ten, that is, the quotient (face / breast) / (face / CGAP) is less than 10 or greater than 0.1.
- the comparison of the subsignificant face / breast data with independent SAGE TM data (face / CGAP) confirms the differential gene expression and validates the markers of the facial skin.
- Table 11 lists further genes that are differentially expressed in facial skin (Face) compared to breast skin (Breast) with a p-value of p ⁇ 0.05 (Signif ⁇ 1, 3) and that in facial skin (Face) im Comparison to other human tissues (except skin), whose expression profiles by the CGAP database are represented, are differentially expressed with a p-value of p> 0.05 (Signif> 1.3), the expression of which differs by more than a power of ten, i.e. the quotient (face / breast) / (Face / CGAP) is greater than 10 or less than 0.1.
- the comparison of the subsignificant face / breast data with independent SAGE TM data (face / CGAP) confirms the differential gene expression and validates the markers of the facial skin.
- Table 12 lists further genes that are differentially expressed in facial skin (Face) compared to breast skin (Breast) with a p-value of p ⁇ 0.05 (Signif ⁇ 1.3) and that in facial skin (Face) im Compared to other human tissues (except skin), whose expression profiles are represented by the CGAP database, are differentially expressed with a p-value of p ⁇ 0.05 (Signif ⁇ 1.3), the expression of which is more than a power of ten differs, that is, the quotient (Face / Breast) / (Face / CGAP) is greater than 10 or less than 0.1.
- the second object underlying the present invention is achieved according to the invention by a method (2) for determining the homeostasis of human facial skin, in particular female facial skin, in vitro, which is characterized in that a) a mixture of proteins, mRNA molecules or fragments of B) the obtained mixture is examined for the presence and possibly the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which are determined by means of serial analysis of gene expression (SAGE) identified as differentially expressed in human facial skin and in other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin, c) comparing the test results from b) with the expression patterns identified by means of serial analysis of gene expression (SAGE) and d) that in b) below requested mixture of healthy or facial skin located in homeostasis if it contains predominantly proteins, mRNA molecules or fragments of proteins or mRNA molecules that are in facial skin are expressed more strongly than in other human tissues (not facial skin), especially in skin-protected areas,
- the mixture in step a) of the method according to the invention for determining the homeostasis of human facial skin can be obtained from whole skin samples, skin equivalents, or cells from human facial skin.
- step b) of the method for determining the homeostasis of human facial 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 facial skin and in other human tissues (not facial skin), especially in skin-protected areas, preferably in breast skin, if these are exclusively in facial skin or exclusively in other human tissues (not facial skin), especially in skin-protected areas , preferably expressed in breast skin. In all other cases, the amount of differentially expressed molecules must also be examined in step b). that is, expression must be quantified.
- SAGE serial analysis of the gene expression
- step d) of the method for determining the homeostasis of human facial skin the mixture of healthy human facial skin examined in b) is assigned if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed more strongly in human facial skin than in other human tissues (not facial skin), especially in skin-protected areas, preferably in breast skin, ie that the mixture either contains more different compounds typically expressed in human facial skin than those that are typically expressed in other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin (qualitative differentiation), or more copies of typically in human Compounds expressed on facial skin contain, than are typically present in other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin (quantitative differentiation).
- the assignment is complementary to the assignment of the human facial skin to sick or disturbed homeostasis.
- a preferred embodiment of the method according to the invention for determining the homeostasis of human facial 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) assigns the mixture of healthy human facial skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed more strongly in healthy human facial skin than in other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin, or that in b) examined mixture of facial skin or skin in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are preferred in other human tissues (not facial skin
- a further preferred embodiment of the method according to the invention for determining the homeostasis of human facial 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 9 and 10 in column 7 by their UniGene Accession Number, in step c) compares the test results from b) with the expression quotients given in Tables 9 and 10 in column 3 and column 5 and in Step d) assigns the mixture of healthy human facial skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed in healthy human facial skin at least 1.9 times as strongly as in others human tissues (not facial skin), especially in skin-protected areas, preferably ise in breast skin, or the mixture examined in b) of diseased or facial skin in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are present
- step b) Determination of the homeostasis of human facial 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 Tables 7 and 8 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) the mixture examined in b) is more healthy human facial skin if it is predominantly proteins, mRNA molecules or fragments of
- Contains proteins or mRNA molecules found in healthy human facial skin are expressed at least 3 times as strongly as in other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin, or the mixture of diseased facial skin or facial skin located in disturbed homeostasis, if it is predominantly proteins, Contains mRNA molecules or fragments of proteins or mRNA molecules which are expressed in other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin, at least 3 times as strongly as in human facial skin.
- a particularly preferred embodiment of the method according to the invention for determining the homeostasis of human facial 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 5 and 6 in column 7 by their UniGene Accession Number, in step c) compares the test results from b) with the expression quotients given in Tables 5 and 6 in column 3 and column 5 and in Step d) assigns the mixture of healthy human facial 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 human facial skin as in other human tissues (not facial skin), especially in skin-protected areas, preferably e in breast skin, or the mixture examined in b) of diseased or facial skin in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are present in
- step b) the mixture obtained is examined for the presence and possibly the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which are defined in Table 4 in column 5 by their UniGene accession number
- step c) compares the test results from b) with the expression quotient given in table 5 in column 3 and in step d) assigns the mixture of healthy human facial skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA Contains molecules that are expressed in healthy human facial skin at least 1.9 times as strongly as in other human tissues (not facial skin), especially in skin-protected areas, preferably in breast skin, or the mixture examined in b) of sick or in attributed to disturbed homeostasis of the facial skin if it is predominantly proteins, mRNA molecules or fragments of proteins or contains mRNA molecules which are expressed in other human tissues (not facial skin),
- a method for determining the homeostasis of human facial 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 5 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 in step d) assigning the mixture of healthy human facial skin examined in b) if it predominantly proteins, mRNA molecules or fragments of
- Tissues (not facial skin), especially in skin-protected areas, preferably in breast skin, or the mixture examined in b) of sick or in assigned to disturbed homeostasis of the facial 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 other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin become like in human facial skin.
- step b) Another particularly preferred embodiment of the method according to the invention for determining the homeostasis of human facial 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 2 in column 5 by their UniGene Accession Number, in step c) comparing the test results from b) with the expression quotients given in table 2 in column 3 and in step d) that examined in b)
- Mixture of healthy human facial 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 healthy human facial skin as in other human tissues (not facial skin), in particular in skin protected areas, preferably in the breast skin, or that in b) classifies the examined mixture of diseased skin or facial skin in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which
- 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 which are defined in Table 1 in column 5 by their UniGene accession number, in step c) comparing the test results from b) with the expression quotients given in table 1 in column 3 and in step d)
- the mixture examined in b) is assigned to healthy human facial skin if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are expressed in healthy human facial skin at least 10 times as strongly as in other human tissues (not facial skin ), especially in skin-protected areas, preferably in breast skin, or the mixture of diseased facial skin or facial skin located in disturbed homeostasis, if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are found in others human tissues (not facial skin), especially in skin-protect
- condition of the facial skin can also be described by quantifying several markers (expression products of the genes which are important for facial skin), which then have to be active in a characteristic relationship to one another in order to represent healthy (in homeostasis) facial skin, or in one different characteristic ratios must be active in order to represent diseased (in disturbed homeostasis) facial skin.
- the present invention therefore furthermore relates to a method (3) for determining the homeostasis of the facial skin in humans, 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 facial 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 facial skin by method (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 and the expression quotient is formed, d) the expression ratios from c) are compared with the expression ratios which are typically found in facial skin for the molecules quantified in b) or in other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin, in particular with the expression ratios that can be found in Tables 1 to 4, Column 3 and Tables 5 to 12, Columns 3 and 5 and e) assign
- the mixture is preferably obtained from a skin sample, in particular from a whole skin sample or from an epidermis sample.
- a 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.
- the mixture is obtained in step a) by means of microdialysis.
- 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. Sei., 1998, 91, 231-244; and also on the Internet at http://www.microdialysis.se/techniqu.htm, which is hereby fully described Scope is referred to.
- microdialysis 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 human facial 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
- MALDI Metal-Assisted Laser Desorption Detection Desorption
- a further preferred embodiment of the method according to the invention for determining the homeostasis of human facial 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) performs the quantification of at least two mRNA molecules or mRNA molecule fragments by means of a method which is selected from Northern blots,
- RT-PCR Reverse transcriptase polymerase chain reaction
- RNase protection experiments iv. Dot blots
- v. cDNA sequencing vi. Clone hybridization
- vii. Differential display viii. Subtractive hybridization
- TOGA Total Gene Expression Analysis
- SAGE Serial analysis of gene expression
- MPSS ® Massively Parallel Signature Sequencing
- the MPSS ® process is US Patent No. 6,013,445 described in, which reference is hereby made in its entirety.
- a further preferred embodiment of the method according to the invention for determining the homeostasis of human facial 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 which are examined in the Tables 1 to 4 in column 5 and Tables 5 to 12 in column 7 are defined by their UniGene Accession Number.
- Another object of the present invention is a test kit for determining the homeostasis of facial skin in humans in vitro, comprising means for carrying out the method according to the invention for determining the homeostasis of human facial skin.
- Another object of the present invention is a biochip for determining the homeostasis of the facial 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, in Tables 1 to 4 in column 5 and in Tables 5 to 12 in column 7 their UniGene Accession Number are defined, especially those defined in Tables 1 to 4 in Column 5 by the following UniGene Accession Numbers:
- Table 2 Hs.344027, Hs.245188, Hs.77910, Hs.77060, Hs.75318, Hs.74304, Hs.3416, Hs.18420, Hs.334305, Hs.287820, Hs.117938;
- 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).
- 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 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 used. 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 (eg 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.
- 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.
- existing DNA molecules can also be bound to surfaces of support material.
- 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 e.g. as described in EP-A-0 965 647:
- the generation of DNA probes takes place here by means of PCR using a sequence-specific primer pair, a primer being modified at the 5 'end and carrying a linker with a free amino group. 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.
- mRNA is isolated from two cell populations to be compared.
- the isolated mRNAs are reverse transcribed using e.g. fluorescence-labeled nucleotides converted into cDNA.
- the samples to be compared are e.g. marked with 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.
- biochips analysis chips mentioned in DE-A-100 28 257.1-52 and in DE-A-101 02 063.5-52 are particularly preferred.
- 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.
- 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.
- the biochip according to the invention comprises peptide or protein probes, in particular antibodies.
- the present invention furthermore relates to the use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which are listed in Tables 1 to 4 in column 5 and in Tables 5 to 12 in column 7 by their UniGene accession. Number can be defined as markers of facial skin in humans.
- Another object of the present invention is a test method for the detection of the effectiveness of cosmetic or pharmaceutical active substances against diseases or impairments of the homeostasis of human facial skin in vitro, characterized in that a) the skin status of human facial skin is determined by an inventive method for determining the homeostasis of human facial skin , or by means of a test kit according to the invention for determining the homeostasis of human facial skin, or by means of a biochip according to the invention, b) applying an active ingredient against diseases or impairments of the homeostasis of human facial skin once or several times to the facial skin, c) again the skin status of human facial skin a method according to the invention for determining the homeostasis of human facial skin, or by means of a test kit according to the invention for determining the homeostasis of human facial skin, or by means of a bioc according to the invention hips determined, and d) the effectiveness of the active ingredient by comparing the results from a) and c) determined.
- test method according to the invention can be carried out with whole skin samples, skin equivalents or cells of human facial 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 human facial 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 in Tables 1 to 4 in column 5 and in Tables 5 to 12 in column 7 defined by their UniGene Accession Number, to demonstrate the effectiveness of cosmetic or pharmaceutical agents against diseases or impairments of the homeostasis of human facial skin.
- Diseases or impairments of the homeostasis of human facial skin include, in particular, pathological conditions of the skin, such as neurodermatitis, sunburn, psoriasis, scleroderma, ichthyosis, atopic dermatitis, acne, seborrhea, lupus erythematosus, rosacea, melanoma, basalioma, skin carcinoma, skin sarcoma, vitamine sarcoma, vitamins sarcoma, vitamins Oily / dry face skin.
- pathological conditions of the skin such as neurodermatitis, sunburn, psoriasis, scleroderma, ichthyosis, atopic dermatitis, acne, seborrhea, lupus erythematosus, rosacea, melanoma, basalioma, skin carcinoma, skin sarcoma, vitamine sarcoma, vitamins sarcoma, vitamins Oily /
- the present invention furthermore relates to a screening method for identifying cosmetic or pharmaceutical active substances against diseases or impairments of the homeostasis of human facial skin in vitro, which is characterized in that a) the skin status of human facial skin is determined by an inventive method for determining homeostasis human facial skin, or by means of a test kit according to the invention for determining the homeostasis of human facial skin, or determined by means of a biochip according to the invention, b) applying a potential active substance against diseases or impairments of the homeostasis of human facial skin to the skin one or more times, c) the human skin status Facial skin using a method according to the invention for determining the homeostasis of human facial skin, or by means of a test kit according to the invention for determining the homeostasis of human facial skin, or using a bi sidesps determined, and d) active ingredients 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 1 to 4 in column 5 and in Tables 5 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 human facial 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 human facial skin, 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 active pharmaceutical ingredients against diseases or impairments of the homeostasis of human facial skin and b) active ingredients found to be effective mixed with cosmetically and pharmacologically suitable and compatible carriers.
Abstract
The invention relates to a method for determining the homeostasis of human facial skin in vitro, to test kits and biochips for determining markers of human facial skin, in addition to the use of proteins, mRNA molecules or fragments of proteins or mRNA molecules as markers of human facial skin. The invention also relates to a test method for detecting the effectiveness of cosmetic or pharmaceutical active substances for treating human facial skin in addition to a screening method for identifying cosmetic or pharmaceutical active substances for treating human facial skin and to a method for producing a cosmetic or pharmaceutical preparation for treating human facial skin.
Description
Verfahren zur Bestimmung von Markern humaner Gesichtshaut Method for the determination of markers of human facial skin
Die vorliegende Erfindung betrifft ein Verfahren zur Bestimmung von Markern humaner Gesichtshaut in vitro, Test-Kits und Biochips zur Bestimmung von Markern humaner Gesichtshaut sowie die Verwendung von Proteinen, mRNA- Molekülen oder Fragmenten von Proteinen oder mRNA-Molekülen als Marker humaner Gesichtshaut; ferner ein Testverfahren zum Nachweis der Wirksamkeit von kosmetischen oder pharmazeutischen Wirkstoffen zur Behandlung humaner Gesichtshaut sowie ein Screening-Verfahren zur Identifikation von kosmetischen oder pharmazeutischen Wirkstoffen zur Behandlung humaner Gesichtshaut und ein Verfahren zur Herstellung einer kosmetischen oder pharmazeutischen Zubereitung zur Behandlung humaner Gesichtshaut.The present invention relates to a method for determining markers of human facial skin in vitro, test kits and biochips for determining markers of human facial skin and the use of proteins, mRNA molecules or fragments of proteins or mRNA molecules as markers of human facial skin; furthermore a test method for the detection of the effectiveness of cosmetic or pharmaceutical active substances for the treatment of human facial skin as well as a screening method for the identification of cosmetic or pharmaceutical active substances for the treatment of human facial skin and a method for the production of a cosmetic or pharmaceutical preparation for the treatment of human facial skin.
Jede lebende Zelle ist in der Lage auf Signale ihrer Umwelt zu reagieren. Die Reaktionen der Zellen werden durch eine geordnete Regulation der Genexpression realisiert, sodaß der Metabolismus von Zellen nicht statisch sondern sehr dynamisch ist. Das menschliche Genom umfasst nach jüngsten Schätzungen zwischen 30 000 und 140.000 Gene. Von diesem immensen Informationsangebot verwendet jede Zelle jedoch lediglich einen kleinen, für sie spezifischen Teil für die Synthese von Proteinen, der sich im Genexpressionsmuster wiederspiegelt. Exogene Signale werden von Zellen empfangen und führen, zum Teil über komplexe Signaltransduktionskaskaden, zu Veränderungen im Genexpressionsmuster. Auf diese Weise reagiert jede Zelle auf Signale aus ihrer Umgebung mit der Anpassung ihres Metabolismus.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.
Neben dieser verhältnismäßig kurzfristigen Veränderung der Genexpression, unterliegt jede lebende Zelle dem Alterungsphänomen, ein Prozess, der mit der langsamer Veränderung der Genexpression einhergeht.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.
Die menschliche Haut ist das größte Organ des menschlichen Körpers. Sie ist ein sehr komplex aufgebautes Organ, welches aus einer Vielzahl verschiedener
Zelltypen besteht und die Grenzfläche des Körpers zur Umwelt bildet. Die meisten Zellen der Haut finden sich in der Epidermis und der Dermis.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.
Hautanhangsgebilde wie z.B. Haarfollikel, Talgdrüsen, Schweissdrüsen etc. werden durch einen geringeren Anteil spezialisierter Zellen gebildet. So sind z.B. nur weniger als 5% der Hautzellen an der Haarfollikelstruktur beteiligt. Diese Tatsache verdeutlicht, dass die Zellen bestimmter Hautanhangsgebilde nur schwer biologischen Analysen, wie z.B. Genexpressionsanalysen unterzogen werden können. Für das Verständnis von Reaktionen der Haut und insbesondere ihrer Anhangsgebilde auf exogene Stimuli ist jedoch die Analyse der Genexpression von entscheidender Bedeutung.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.
Eine Isolation der Hautanhangsgebilde ist technisch schwer zu realisieren und sehr zeitintesiv. Desweiteren ist es für eine realistische Darstellung biochemischer Abläufe in der Haut oder ihrer Anhangsgebilde unerlässlich, die Zellen in ihrer natürlichen, zellulären Umgebung zu untersuchen. Jede Manipulation am Gewebe (z.B. zur Isolation oder Anreicherung bestimmter Strukturen) wird von den Zellen bemerkt und führt zu einer angepassten Genexpression. Dieser Zustand ist nicht mehr nativ und somit auch nicht mehr als repräsentativ zu betrachten.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.
Die menschliche Gesichtshaut ist kontinuierlich der Umwelt und vielfältigen Stressoren ausgesetzt. Es ist daher zu erwarten, dass insbesondere die ungeschützte Gesichtshaut auf die diversen Stressoren der Umwelt reagiert. Welche molekularen Mechanismen diesen Reaktionen zugrunde liegen ist bislang weitgehend unklar. Effektive kosmetische oder pharmazeutische Produkte für die Gesichtshaut sollten ihre positive Wirkung auf ein möglichst breites Spektrum molekularer Abläufe im Gewebe zeigen. Bisher sind jedoch nur wenige molekulare Reaktionsmechanismen in Gesichtshaut beschrieben worden, die somit als Target z.B. für kosmetische Gesichts-Produkte dienen können.The human face skin is continuously exposed to the environment and various stressors. It is therefore to be expected that especially the unprotected facial skin will react to the various environmental stressors. So far it is largely unclear which molecular mechanisms underlie these reactions. Effective cosmetic or pharmaceutical products for the facial skin should have a positive effect on the broadest possible range of molecular processes in the tissue. So far, however, only a few molecular reaction mechanisms in facial skin have been described, which are thus targeted e.g. can be used for cosmetic facial products.
Jeder Zelltyp der Haut und ihrer Anhangsgebilde exprimiert ca. 15.000 verschiedene Gene und synthetisiert daraus entsprechend viele Proteine. Welche
Gene davon in Gesichtshaut eine Rolle spielen ist bisher jedoch weitgehend unklar.Each cell type of the skin and its appendages express approximately 15,000 different genes and synthesize a corresponding number of proteins from them. Which Genes of which play a role in facial skin have so far been largely unclear.
Die Haut besteht aus mehreren verschiedenen Zelltypen (z. B. aus Fibroblasten, Keratinozyten in verschiedenen Differenzierungszuständen, Melanozyten, Merkelzellen, Langerhanszellen, einer Vielzahl unterscheidlicher Zellen des Haarfollikels oder anderer Hautanhangsgebilde), sodass die Komplexität in der Haut exprimierter Gene sehr groß ist. Es ist bisher nicht möglich gewesen, aus dieser Komplexität die Gene zu identifizieren, die mit der Gesichtshaut in Zusammenhang stehen und als molekulare Marker dieses Gewebes dienen können. Erschwerend kommt hinzu, dass in der Zelle mRNA-Moleküle in Konzentrationen zwischen einigen wenigen und mehreren hundert Kopien vorkommen. Die schwach exprimierten Gene sind bisherigen Analysetechniken nicht oder nur sehr schwer zugänglich gewesen, können aber durchaus eine entscheidende Rolle in der Gesichtshaut spielen.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. It has so far not been possible to identify from this complexity the genes which are associated with the facial skin and which can serve as molecular markers of this tissue. 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 for analysis techniques or have been very difficult to access, but they can certainly play a decisive role in the facial skin.
Bis heute ist das Transkriptom, also die Gesamtheit aller transkribierten Gene humaner Gesichtshaut, nicht beschrieben worden.The transcriptome, i.e. the entirety of all transcribed genes of human facial skin, has not been described to date.
Transkriptom-Analysen der Haut mittels verschiedener Verfahren, einschließlich der SAGE™-Analyse, sind Stand der Technik. Allerdings werden hierbei isolierte Keratinozyten (in vitro) oder Epidermis-Explantate verwendet, die - wie oben erläutert - keine für das komplexe Geschehen in der Haut repräsentativen Modelle darstellen.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 explained above - do not represent models representative of the complex process in the skin.
Aus der DE-A-101 00 127.4-41 der Anmelderin ist bekannt, Hautzellen einer SAGE™-Analyse zu untenwerfen, um das Gesamttranskriptom der Haut zu charakterisieren. Die DE-A-101 00 121.5-41 der Anmelderin offenbart die Ermittlung Von Markern gestreßter bzw. gealterter Haut auf der Basis einer vergleichenden SAGE™-Analyse zwischen gestreßter bzw. gealterter Haut und ungestreßter bzw. junger Haut. Informationen über spezifische Marker humaner Gesichtshaut sind diesen Schriften aber nicht zu entnehmen.
Aus J Invest Dermatol 2002 Jul;119(1):3-13; „A serial analysis of gene expression in sun-damaged human skin"; Urschitz J. et al.; ist bekannt, Marker sonnengeschädigter Haut mittels einer vergleichenden SAGE™-Analyse von Vollhautexplantanten zu bestimmen, die vor der Ohrmuschel (sonnengeschädigt) bzw. hinter der Ohrmuschel (geschützt vor Sonnenstrahlung) entnommen wurden. Aus dieser Publikation lassen sich gleichfalls keinerlei Kenntnisse über spezifische Marker humaner Gesichtshaut gewinnen.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 human facial 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 of human facial skin can be gained from this publication.
Es besteht daher ein Bedarf an der Identifikation möglichst vieler, vorzugsweise aller, für die humane Gesichtshaut wichtigen Gene, insbesondere der für die humane Gesichtshaut spezifisch wichtigen Gene.There is therefore a need to identify as many, preferably all, of the genes that are important for the human facial skin, especially the genes that are specifically important for the human facial skin.
Aufgabe der vorliegenden Erfindung ist es daher, einen möglichst großen Teil der für die humane Gesichtshaut bedeutsamen Gene zu identifizieren. Außerdem sollen mittels der identifizierten Gene Verfahren zur Bestimmung der Homeostase humaner Gesichtshaut bereitgestellt werden.The object of the present invention is therefore to identify as large as possible a part of the genes which are important for the human facial skin. The identified genes are also intended to provide methods for determining the homeostasis of human facial skin.
Diese erste Aufgabe wird erfindungsgemäß gelöst durch ein Verfahren (1) zur Identifizierung der für die humane Gesichtshaut bedeutsamen Gene bei Menschen in vitro, dadurch gekennzeichnet, daß man a) ein erstes Gemisch von in humaner Gesichtshaut exprimierten, d. h. transkribierten und gegebenenfalls auch translatierten genetisch codierten Faktoren, also von Proteinen, mRNA-Molekülen oder Fragmenten von Proteinen oder mRNA-Molekülen aus humaner Gesichtshaut gewinnt, b) ein zweites Gemisch von in anderen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut, exprimierten, d. h. transkribierten und gegebenenfalls auch translatierten genetisch codierten Faktoren, also von Proteinen, mRNA- Molekülen oder Fragmenten von Proteinen oder mRNA-Molekülen aus anderen menschlichen Geweben (nicht Gesichtshaut), insbesondere aus Haut geschützter Areale, vorzugsweise aus Brusthaut, gewinnt und
c) die in a) und b) gewonnenen Gemische einer Seriellen Analyse der Genexpression (SAGE) unterwirft, und dadurch die Gene identifiziert, die in humaner Gesichtshaut unterschiedlich stark (differentiell) exprimiert werden.This first object is achieved according to the invention by a method (1) for identifying the genes which are important for human facial skin in humans in vitro, characterized in that a) a first mixture of genetically coded, ie transcribed and optionally also translated, expressed in human facial skin Factors, ie proteins, mRNA molecules or fragments of proteins or mRNA molecules from human facial skin, b) a second mixture of areas expressed in other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin, ie expressed transcribed and possibly also translated genetically coded factors, that is, proteins, mRNA molecules or fragments of proteins or mRNA molecules from other human tissues (not facial skin), in particular from skin-protected areas, preferably from breast 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 human facial skin.
Durch das erfindungsgemäße Verfahren wird es vorteilhafterweise möglich, den komplexen Prozess zellulärer Reaktionen auf die Umwelt und die zu Grundeliegenden kausalen Zusammenhänge in der Gesichtshaut zu begreifen. Mit diesem Wissen können neue Konzepte für kosmetische Gesichts-Produkte entwickelt werden, die ihre Wirkung auf das breite Spektrum der Genexpression in Gesichtshaut ausüben. Die im Rahmen des erfindungsgemäßen Verfahrens durchgeführte SAGE™-Analyse zeigt erstmals, welche Gene in Gesichtshaut exprimiert werden, und welche Gene dort anders exprimiert werden als in anderen Geweben, insbesondere in der Haut geschützter Areale, wie z.B. der Brusthaut.The method according to the invention advantageously makes it possible to understand the complex process of cellular reactions to the environment and the underlying causal relationships in the facial skin. With this knowledge, new concepts for cosmetic facial products can be developed that have an effect on the broad spectrum of gene expression in facial skin. The SAGE ™ analysis carried out as part of the method according to the invention shows for the first time which genes are expressed in facial skin and which genes are expressed there differently than in other tissues, in particular in the skin of protected areas, such as e.g. the breast skin.
Die am Markt befindlichen kosmetischen Gesichts-Produkte üben ihre Wirkungen zumeist auf einige wenige bekannte Marker der Haut aus (z.B. Kollagen). Erst die vorliegenden Untersuchungsergebnisse erlauben ein Verständnis der komplexen biologischen Prozesse im humaner Gesichtshaut. Die Identifikation geeigneter Marker der Gesichtshaut gestattet somit die gezielte Suche nach Substanzen oder Kombinationen von Substanzen mit einem breiten Wirkspektrum auf die Genexpression in Gesichtsgewebe. Produkte dieser Art konnten jedoch bis zu dem jetzigen Zeitpunkt nicht entwickelt werden, da eine Vielzahl der Gesichtshautmarker noch nicht bekannt waren.The cosmetic facial products on the market mostly exert their effects on a few known markers of the skin (e.g. collagen). Only the available investigation results allow an understanding of the complex biological processes in the human facial skin. The identification of suitable markers of the facial skin thus enables the targeted search for substances or combinations of substances with a broad spectrum of action on gene expression in facial tissue. However, products of this type have not yet been able to be developed because a large number of facial skin markers were not yet known.
Die Gesamtheit aller m-RNA-Moleküle, die von einer Zelle oder einem Gewebe zu einem bestimmten Zeitpunkt synthetisiert werden, bezeichnet man als "Transkriptom". Zur Erfassung des Transkriptoms der Gesichtshaut wurde die Technik der „Seriellen Analyse der Genexpression" (SAGE™) eingesetzt. Diese Technik erlaubt gleichzeitig die Identifikation und Quantifizierung aller in der Gesichtshaut exprimierten Gene. Die Analyse der Genexpression ist zwar auch mit der Quantifizierung spezifischer mRNA-Moleküle möglich (z.B. Northem-Blot, RNase-Schutzexperimente). Mit diesen Techniken können jedoch nur eine relativ begrenzte Anzahl an Genen gemessen werden. Theoretisch könnten die
Techniken MPSS (Massive Parallel Signiture Sequencing) oder Techniken, die auf Differential display beruhen, die SAGE™-Analyse ersetzen. Praktisch ist die SAGE™-Technik jedoch bislang schneller und zuverlässiger als Alternativmethoden und somit zu bevorzugen.The entirety of all m-RNA molecules that are synthesized by a cell or a tissue at a specific point in time is referred to as a "transcriptome". The technique of "serial analysis of gene expression" (SAGE ™) was used to record the transcriptome of the facial skin. This technique simultaneously allows the identification and quantification of all genes expressed in the facial skin. The analysis of the gene expression is also possible with the quantification of specific mRNA Molecules are possible (eg Northem blot, RNase protection experiments), but these techniques can only measure a relatively limited number of genes Techniques MPSS (Massive Parallel Signiture Sequencing) or techniques based on differential display that replace SAGE ™ analysis. In practice, however, the SAGE ™ technology has so far been faster and more reliable than alternative methods and is therefore preferable.
Der Vergleich des Transkriptoms humaner Gesichtshaut, mit dem Transkriptom anderer menschlicher Gewebe (nicht Gesichtshaut), insbesondere der Haut geschützter Areale, vorzugsweise der Brusthaut, lässt die Unterscheidung zwischen für die Gesichtshaut relevanten und nicht relevanten Genen zu. Dies können Gene sein, die in Gesichtshaut besonders stark exprimiert werden oder auch Gene sein, die dadurch gekennzeichnet sind, dass sie im Vergleich zur Brusthaut nur gering exprimiert werden.The comparison of the transcriptome of human facial skin with the transcriptome of other human tissues (not facial skin), in particular the skin of protected areas, preferably the breast skin, allows the distinction between genes relevant and irrelevant to the facial skin. These can be genes that are particularly strongly expressed in facial skin or genes that are characterized in that they are only expressed slightly compared to the breast skin.
Im Rahmen von plastisch chirurgischen Operationen wie z.B. „Unteren Facelifts" oder „Mammareduktionen" fallen von Patienten Gewebe aus dem Bereich vor dem Ohr (Gesichtshaut) und auch von der weiblichen Brust an. Die Analyse solcher Gewebeproben erlaubt daher die Beschreibung der Transkriptome der Gesichtshaut und der Brusthaut. Der Vergleich beider Transkriptome zeigt welche Gene besonders berücksichtigt werden müssen.In the context of plastic surgical operations such as "Lower facelifts" or "breast reductions" occur in patients from the area in front of the ear (facial skin) and also from the female breast. The analysis of such tissue samples therefore allows the description of the transcriptomes of the facial skin and the breast skin. The comparison of both transcriptomes shows which genes need special attention.
Nach seriösen Schätzungen beträgt der Anteil an mRNA-Spezies die in maximal 5 Kopien pro Zelle vorliegen rund 87%. Dieser Anteil gering exprimierter Gene läßt vermuten, dass die Expression Gesichtshaut-spezifischer Gene ebenfalls zu rund 87% durch gering exprimierte Gene dargestellt sein könnte.According to serious estimates, the proportion of mRNA species that are present in a maximum of 5 copies per cell is around 87%. This proportion of low-expressed genes suggests that the expression of facial skin-specific genes could also be represented by around 87% by low-expressed genes.
Um die Gesichtshaut-spezifische Genexpression dennoch zu erfassen, können auch geringe, statistisch wenig signifikante Unterschiede durch weitere relevante SAGE™ -Analysen mit in die Auswertung aufgenommen werden. So können z.b. auf der Basis von SAGE™ -Analysen ermittelte Genexpressionsdaten mit einer öffentlich zugänglichen SAGE™-Datenbank (CGAP), die aus ca. 2.5 Mio Tags besteht, die alle nicht aus Haut stammen, verglichen und so gesichert werden. Die CGAP-Datenbank ist beim NCBI online unter der URL http://www.ncbi.nlm.nih.gov/SAGE einsehbar.
Der vorliegenden Anmeldung liegt die CGAP-Datenbank in einer Version vomIn order to record facial skin-specific gene expression, even small, statistically insignificant differences can be included in the evaluation by means of further relevant SAGE ™ analyzes. For example, gene expression data determined on the basis of SAGE ™ analyzes can be compared with a publicly accessible SAGE ™ database (CGAP), which consists of approx. 2.5 million tags, all of which do not come from the skin. The CGAP database can be viewed online at the NCBI at the URL http://www.ncbi.nlm.nih.gov/SAGE. The present application has the CGAP database in a version dated
24.09.2000 zugrunde. Im einzelnen umfasst die Datenbank 66 Projekte, die von der URL ftp://ftp.ncbi.nlm.nih.gov/pub/sage/fasta erhalten wurden.24.09.2000 based. In detail, the database contains 66 projects that were obtained from the URL ftp://ftp.ncbi.nlm.nih.gov/pub/sage/fasta.
Nachfolgend sind die Bezeichnungen der Projekte und ihr letzterBelow are the names of the projects and their last
Aktualisierungsstand angegeben:Update status given:
Sep 52000 Duke_H247_HypoxiaSep 52000 Duke_H247_Hypoxia
Aug 142000 pooled_GBMAug 142000 pooled_GBM
Aug 142000 normal_pool_6thAug 142000 normal_pool_6th
Aug 14 2000 normal_cerebellumAug 14 2000 normal_cerebellum
Aug 14 2000 mammary_epitheliumAug 14 2000 mammary epithelium
Aug 142000 Tu98Aug 142000 Tu98
Aug 142000 Tu 102Aug 142000 Tu 102
Aug 142000 TSUAug 142000 TSU
Aug 142000 SciencePark_MCF7_estradiol_10hAug 142000 SciencePark_MCF7_estradiol_10h
Aug 142000 SciencePark_MCF7_estradiol_3hAug 142000 SciencePark_MCF7_estradiol_3h
Aug 142000 SciencePark_MCF7_control_3hAug 142000 SciencePark_MCF7_control_3h
Aug 14 2000 SciencePark_MCF7_Control_0hAug 14 2000 SciencePark_MCF7_Control_0h
Aug 142000 SW837Aug 142000 SW837
Aug 142000 SKBR3Aug 142000 SKBR3
Aug 142000 RKOAug 142000 RKO
Aug 14 2000 Panc_96-6252Aug 14 2000 Panc_96-6252
Aug 142000 Panc_91-16113Aug 142000 Panc_91-16113
Aug 142000 OVT-8Aug 142000 OVT-8
Aug 142000 OVT-7Aug 142000 OVT-7
Aug 14 2000 OVT-6Aug 14 2000 OVT-6
Aug 142000 OVP-5Aug 142000 OVP-5
Aug 142000 OVCA432-2Aug 142000 OVCA432-2
Aug 142000 OV1063-3Aug 142000 OV1063-3
Aug 142000 NHA_5thAug 142000 NHA_5th
Aug 142000 NC2
Aug 142000 NC1Aug 142000 NC2 Aug 142000 NC1
Aug 14 2000 ML10-10Aug 14 2000 ML10-10
Aug 14 2000 MDA453Aug 14 2000 MDA453
Aug 142000 LNCaPAug 142000 LNCaP
Aug 14 2000 IOSE29-11Aug 14 2000 IOSE29-11
Aug 142000 HOSE_4Aug 142000 HOSE_4
Aug 142000 HCT116Aug 142000 HCT116
Aug 14 2000 H1126Aug 14 2000 H1126
Aug 142000 ES2-1Aug 142000 ES2-1
Aug 142000 Duke halamusAug 142000 Duke Halamus
Aug 14 2000 Duke_post_crisis_fibroblastsAug 14 2000 Duke_post_crisis_fibroblasts
Aug 142000 Duke_precrisis_fibroblastsAug 142000 Duke_precrisis_fibroblasts
Aug 142000 Duke_cerebellumAug 142000 Duke_cerebellum
Aug 14 2000 Duke_HMVEC_VEGFAug 14 2000 Duke_HMVEC_VEGF
Aug 14 2000 Duke_HMVECAug 14 2000 Duke_HMVEC
Aug 142000 Duke_H341Aug 142000 Duke_H341
Aug 14 2000 Duke_H247_normalAug 14 2000 Duke_H247_normal
Aug 14 2000 DukeJH 1043Aug 14 2000 DukeJH 1043
Aug 142000 DukeJH 1020Aug 142000 DukeJH 1020
Aug 142000 Duke_BB542_normal__cerebellumAug 142000 Duke_BB542_normal__cerebellum
Aug 14 2000 Duke_GBM_H1110Aug 14 2000 Duke_GBM_H1110
Aug 142000 Duke_96-349Aug 142000 Duke_96-349
Aug 14 2000 Duke_757Aug 14 2000 Duke_757
Aug 142000 Duke_48NAug 142000 Duke_48N
Aug 142000 Duke_40NAug 142000 Duke_40N
Aug 142000 Duke_1273Aug 142000 Duke_1273
Aug 14 2000 Duke-H988Aug 14 2000 Duke-H988
Aug 14 2000 DCISAug 14 2000 DCIS
Aug 14 2000 Caco_2Aug 14 2000 Caco_2
Aug 14 2000 Chen_Tumor_PrAug 14 2000 Chen_Tumor_Pr
Aug 142000 Chen_Normal_PrAug 142000 Chen_Normal_Pr
Aug 142000 Chen_LNCaP_no-DHTAug 142000 Chen_LNCaP_no-DHT
Aug 14 2000 Chen_LNCaP
Aug 142000 CPDR_LNCaP-T Aug 14 2000 CPDR_LNCaP-C Aug 142000 Br_N Aug 142000 BB542_whitematter Aug 14 2000 Aplus Aug 14 2000 A2780-9 Aug 142000293-CTRL Aug 122000 Duke_mhh-1Aug 14 2000 Chen_LNCaP Aug 142000 CPDR_LNCaP-T Aug 14 2000 CPDR_LNCaP-C Aug 142000 Br_N Aug 142000 BB542_whitematter Aug 14 2000 Aplus Aug 14 2000 A2780-9 Aug 142000293-CTRL Aug 122000 Duke_mhh-1
Für die SAGE™-Analyse wurde humane Gesichtshaut von einer gesunden weiblichen Spenderin (65 Jahre alt) und Brusthaut von einer anderen gesunden weiblichen Spenderin (69 Jahre alt) verwendet. Die Durchführung der SAGE™- Analyse erfolgte, wie in der EP-A-0 761 822 und bei Veiculescu, V.E. et al., 1995 Science 270, 484-487, beschrieben. Diese Technik erlaubt gleichzeitig die Identifikation und Quantifizierung der in Gesichtshaut exprimierten Gene.Human facial skin from a healthy female donor (65 years old) and breast skin from another healthy female donor (69 years old) were used for the SAGE ™ analysis. The SAGE ™ analysis was carried out as described in EP-A-0 761 822 and at Veiculescu, V.E. et al., 1995 Science 270, 484-487. This technique also allows the identification and quantification of the genes expressed in facial skin.
Zur bioinformatischen Analyse wurden beide SAGE™-Libraries (Gesichtshaut- Library und Brust-Library) auf die durchschnittliche Tag-Anzahl normiert, miteinander verglichen und Gene mit einer Gesichtshaut-spezifischen Regulation identifiziert. Wie für zwei Libraries desselben Gewebetyps erwartet, ist das Tag- Repertoire der beiden Haut-Libraries weitgehend ähnlich.For bioinformatic analysis, both SAGE ™ libraries (facial skin library and breast library) were normalized to the average number of tags, compared with one another and genes with a facial skin-specific regulation were identified. As expected for two libraries of the same tissue type, the tag repertoire of the two skin libraries is largely similar.
Zur Bestätigung differentiell exprimierter Gene mit einer geringen statistischen Signifikanz, wurden zuzüglich die SAGE™-Daten der Gesichtshaut mit der obengenannten CGAP-Bank verglichen. Da hier zwei SAGE-Banken verglichen wurden, die eine stark unterschiedliche Tag-Häufigkeit aufweisen, wurden nicht beide SAGE™-Libraries auf ihren gemeinsamen Mittelwert normiert, sondern die CGAP-Bank auf die SAGE™-Library der Gesichtshaut normiert.To confirm differentially expressed genes with a low statistical significance, the SAGE ™ data of the facial skin were compared with the above-mentioned CGAP library. Since two SAGE banks with a very different tag frequency were compared here, both SAGE ™ libraries were not standardized to their common mean, but the CGAP bank to the SAGE ™ library of the facial skin.
Die Tabellen 1 bis 4 enthalten eine detaillierte Auflistung der mit Hilfe des erfindungsgemäßen Verfahrens ermittelten, in Gesichtshaut und in anderen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut differentiell exprimierten Gene unter Angabe
• einer laufenden Ordnungsnummer in Spalte 1 ,Tables 1 to 4 contain a detailed list of the genes which are determined with the aid of the method according to the invention and which are differentially expressed in facial skin and in other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin • a serial number in column 1,
• der verwendeten Tag-Sequenz in Spalte 2,The tag sequence used in column 2,
• des Quotienten der ermittelten relativen Expressionsfrequenz in Gesichtshaut (Face) und der ermittelten relativen Expressionsfrequenz in Brusthaut (Breast) in Spalte 3,The quotient of the determined relative expression frequency in facial skin (Face) and the determined relative expression frequency in breast skin (Breast) in column 3,
• der Signifikanz der in Spalte 3 genannten Werte in Spalte 4,The significance of the values mentioned in column 3 in column 4,
• der UniGene-Accession-Number in Spalte 5 und• the UniGene Accession Number in column 5 and
• einer Kurzbeschreibung des Gens bzw. Genproduktes in Spalte 6• a brief description of the gene or gene product in column 6
Die Tabellen 5 bis 12 enthalten eine detaillierte Auflistung der mit Hilfe des erfindungsgemäßen Verfahrens ermittelten, in Gesichtshaut und in anderen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut differentiell exprimierten Gene unter AngabeTables 5 to 12 contain a detailed list of the genes determined with the aid of the method according to the invention and differentially expressed in facial skin and in other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin
• einer laufenden Ordnungsnummer in Spalte 1 ,• a serial number in column 1,
• der verwendeten Tag-Sequenz in Spalte 2,The tag sequence used in column 2,
• des Quotienten der ermittelten relativen Expressionsfrequenz in Gesichtshaut (Face) und der ermittelten relativen Expressionsfrequenz in Brusthaut (Breast) in Spalte 3,The quotient of the determined relative expression frequency in facial skin (Face) and the determined relative expression frequency in breast skin (Breast) in column 3,
• der Signifikanz der in Spalte 3 genannten Werte in Spalte 4,The significance of the values mentioned in column 3 in column 4,
• des Quotienten der ermittelten relativen Expressionsfrequenz in Gesichtshaut (Face) und der relativen Expressionsfrequenz in der CGAP- Datenbank in Spalte 5,The quotient of the determined relative expression frequency in facial skin (face) and the relative expression frequency in the CGAP database in column 5,
• der Signifikanz der in Spalte 5 genannten Werte in Spalte 6,The significance of the values mentioned in column 5 in column 6,
• der UniGene-Accession-Number in Spalte 7,• the UniGene Accession Number in column 7,
• einer Kurzbeschreibung des Gens bzw. Genproduktes in Spalte 8 und• a brief description of the gene or gene product in column 8 and
• des Quotienten (Face/Breast) / (Face/CGAP) in Spalte 9.• the quotient (Face / Breast) / (Face / CGAP) in column 9.
Der Quotient in Spalte 3 gibt die Stärke der differentiellen Expression an, d. h., um welchen Faktor das jeweilige Gen in Gesichtshaut (Face) stärker exprimiert wird, als in Brusthaut (Breast), oder umgekehrt.
Der Quotient in Spalte 5 gibt die Stärke der differentiellen Expression an, d. h., um welchen Faktor das jeweilige Gen in Gesichtshaut (Face) stärker exprimiert wird, als in sonstigen menschlichen Geweben (außer Haut), deren Expressionsprofile durch die CGAP-Datenbank repräsentiert werden, oder umgekehrt.The quotient in column 3 indicates the strength of the differential expression, ie the factor by which the respective gene is expressed more strongly in facial skin (face) than in breast skin (breast), or vice versa. The quotient in column 5 indicates the strength of the differential expression, ie by which factor the respective gene is expressed more strongly in facial skin (face) than in other human tissues (except skin) whose expression profiles are represented by the CGAP database, or the other way around.
Unter ihrer UniGene-Accession-Number sind die jeweiligen Gene bzw. Genprodukte in der Datenbank des National Center for Biotechnology Information (NCBI) offenbart. Diese Datenbank ist im Internet unter folgender Adresse zugänglich: http://www.ncbi.nlm.nih.gov/.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/.
Die Gene bzw. Genprodukte sind außerdem unter den Internet-Adressen http://www.ncbi.nlm.nih.gov/UniGene/Hs.Home.html oder http://www.ncbi.nlm.nih.gov/genome/guide direkt zugänglich.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.
In Tabelle 1 sind alle Gene aufgelistet, die in Gesichtshaut (Face) im Vergleich zu Brusthaut (Breast) mit einem p-Value von p>0,05 (Signif >1,3) mindestens 10-fach differentiell exprimiert werden.Table 1 lists all genes that are at least 10-fold differentially expressed in facial skin (face) compared to breast skin (breast) with a p-value of p> 0.05 (signif> 1.3).
In Tabelle 2 sind alle Gene aufgelistet, die in Gesichtshaut (Face) im Vergleich zu Brusthaut (Breast) mit einem p-Value von p>0,05 (Signif >1 ,3) mindestens 5-fach differentiell exprimiert werden.Table 2 lists all genes that are expressed differentially in facial skin (face) compared to breast skin (breast) with a p-value of p> 0.05 (Signif> 1, 3) at least 5-fold.
In Tabelle 3 sind alle Gene aufgelistet, die in Gesichtshaut (Face) im Vergleich zu Brusthaut (Breast) mit einem p-Value von p>0,05 (Signif >1,3) mindestens 3-fach differentiell exprimiert werden.Table 3 lists all genes that are at least 3 times differentially expressed in facial skin (face) compared to breast skin (breast) with a p-value of p> 0.05 (signif> 1.3).
In Tabelle 4 sind alle Gene aufgelistet, die in Gesichtshaut (Face) im Vergleich zu Brusthaut (Breast) mit einem p-Value von p>0,05 (Signif >1,3) mindestens 1,9- fach differentiell exprimiert werden.Table 4 lists all genes that are at least 1.9 times differentially expressed in facial skin (face) compared to breast skin (breast) with a p-value of p> 0.05 (signif> 1.3).
In Tabelle 5 sind alle Gene aufgelistet, die in Gesichtshaut (Face) im Vergleich zu Brusthaut (Breast) mit einem p-Value von p<0,05 (Signif <1 ,3) mindestens 5-fach differentiell exprimiert werden und die in Gesichtshaut (Face) im Vergleich zu sonstigen menschlichen Geweben (außer Haut), deren Expressionsprofile durch
die CGAP-Datenbank repräsentiert werden, mit einem p-Value von p>0,05 (Signif >1,3) mindestens 5-fach differentiell exprimiert werden. Der Vergleich der subsignifikanten Face/Breast-Daten mit unabhängigen SAGE™-Daten (Face/CGAP) bestätigt die differentielle Genexpression und validiert die Marker der Gesichtshaut.Table 5 lists all genes that are at least 5 times differentially expressed in facial skin (Face) compared to breast skin (Breast) with a p-value of p <0.05 (Signif <1, 3) and those in facial skin (Face) compared to other human tissues (except skin), whose expression profiles by the CGAP database are represented with a p-value of p> 0.05 (Signif> 1.3) are expressed at least 5-fold differentially. The comparison of the subsignificant face / breast data with independent SAGE ™ data (face / CGAP) confirms the differential gene expression and validates the markers of the facial skin.
In Tabelle 6 sind alle Gene aufgelistet, die in Gesichtshaut (Face) im Vergleich zu Brusthaut (Breast) mit einem p-Value von p<0,05 (Signif <1 ,3) mindestens 5-fach differentiell exprimiert werden und die in Gesichtshaut (Face) im Vergleich zu sonstigen menschlichen Geweben (außer Haut), deren Expressionsprofile durch die CGAP-Datenbank repräsentiert werden, mit einem p-Value von p<0,05 (Signif <1 ,3) mindestens 5-fach differentiell exprimiert werden, deren Expression sich um weniger als eine Zehnerpotenz unterscheidet, dh., der Quotient (Face/Breast) / (Face/CGAP) ist kleiner als 10 oder größer als 0,1. Der Vergleich der subsignifikanten Face/Breast-Daten mit unabhängigen SAGE™-Daten (Face/CGAP) bestätigt die differentielle Genexpression und validiert die Marker der Gesichtshaut.Table 6 lists all genes that are expressed differentially in facial skin (face) compared to breast skin (breast) with a p-value of p <0.05 (Signif <1, 3) and that in facial skin (Face) compared to other human tissues (except skin), whose expression profiles are represented by the CGAP database, with a p-value of p <0.05 (Signif <1, 3) are expressed at least 5-fold differentially, whose expression differs by less than a power of ten, i.e. the quotient (Face / Breast) / (Face / CGAP) is less than 10 or greater than 0.1. The comparison of the subsignificant face / breast data with independent SAGE ™ data (face / CGAP) confirms the differential gene expression and validates the markers of the facial skin.
In Tabelle 7 sind alle Gene aufgelistet, die in Gesichtshaut (Face) im Vergleich zu Brusthaut (Breast) mit einem p-Value von p<0,05 (Signif <1 ,3) mindestens 3-fach differentiell exprimiert werden und die in Gesichtshaut (Face) im Vergleich zu sonstigen menschlichen Geweben (außer Haut), deren Expressionsprofile durch die CGAP-Datenbank repräsentiert werden, mit einem p-Value von p>0,05 (Signif >1 ,3) mindestens 3-fach differentiell exprimiert werden. Der Vergleich der subsignifikanten Face/Breast-Daten mit unabhängigen SAGE™-Daten (Face/CGAP) bestätigt die differentielle Genexpression und validiert die Marker der Gesichtshaut.Table 7 lists all genes that are at least 3 times differentially expressed in facial skin (Face) compared to breast skin (Breast) with a p-value of p <0.05 (Signif <1, 3) and those in facial skin (Face) compared to other human tissues (except skin), whose expression profiles are represented by the CGAP database, with a p-value of p> 0.05 (Signif> 1, 3) are expressed at least 3-fold differentially. The comparison of the subsignificant face / breast data with independent SAGE ™ data (face / CGAP) confirms the differential gene expression and validates the markers of the facial skin.
In Tabelle 8 sind alle Gene aufgelistet, die in Gesichtshaut (Face) im Vergleich zu Brusthaut (Breast) mit einem p-Value von p<0,05 (Signif <1 ,3) mindestens 3-fach differentiell exprimiert werden und die in Gesichtshaut (Face) im Vergleich zu sonstigen menschlichen Geweben (außer Haut), deren Expressionsprofile durch die CGAP-Datenbank repräsentiert werden, mit einem p-Value von p<0,05 (Signif
<1,3) mindestens 3-fach differentiell exprimiert werden, deren Expression sich um weniger als eine Zehnerpotenz unterscheidet, dh., der Quotient (Face/Breast) / (Face/CGAP) ist kleiner als 10 oder größer als 0,1. Der Vergleich der subsignifikanten Face/Breast-Daten mit unabhängigen SAGE™-Daten (Face/CGAP) bestätigt die differentielle Genexpression und validiert die Marker der Gesichtshaut.Table 8 lists all genes that are at least 3 times differentially expressed in facial skin (Face) compared to breast skin (Breast) with a p-value of p <0.05 (Signif <1, 3) and that in facial skin (Face) compared to other human tissues (except skin), whose expression profiles are represented by the CGAP database, with a p-value of p <0.05 (signif <1.3) are expressed differentially at least three times, the expression of which differs by less than a power of ten, i.e. the quotient (face / breast) / (face / CGAP) is less than 10 or greater than 0.1. The comparison of the subsignificant face / breast data with independent SAGE ™ data (face / CGAP) confirms the differential gene expression and validates the markers of the facial skin.
In Tabelle 9 sind alle Gene aufgelistet, die in Gesichtshaut (Face) im Vergleich zu Brusthaut (Breast) mit einem p-Value von p<0,05 (Signif <1,3) mindestens 1,9- fach differentiell exprimiert werden und die in Gesichtshaut (Face) im Vergleich zu sonstigen menschlichen Geweben (außer Haut), deren Expressionsprofile durch die CGAP-Datenbank repräsentiert werden, mit einem p-Value von p>0,05 (Signif >1,3) mindestens 1 ,9-fach differentiell exprimiert werden. Der Vergleich der subsignifikanten Face/Breast-Daten mit unabhängigen SAGE™-Daten (Face/CGAP) bestätigt die differentielle Genexpression und validiert die Marker der Gesichtshaut.Table 9 lists all genes which are expressed differentially in facial skin (Face) compared to breast skin (Breast) with a p-value of p <0.05 (Signif <1.3) and which in facial skin (face) compared to other human tissues (except skin), whose expression profiles are represented by the CGAP database, with a p-value of p> 0.05 (Signif> 1.3) at least 1.9 times be differentially expressed. The comparison of the subsignificant face / breast data with independent SAGE ™ data (face / CGAP) confirms the differential gene expression and validates the markers of the facial skin.
In Tabelle 10 sind alle Gene aufgelistet, die in Gesichtshaut (Face) im Vergleich zu Brusthaut (Breast) mit einem p-Value von p<0,05 (Signif <1,3) mindestens 1,9- fach differentiell exprimiert werden und die in Gesichtshaut (Face) im Vergleich zu sonstigen menschlichen Geweben (außer Haut), deren Expressionsprofile durch die CGAP-Datenbank repräsentiert werden, mit einem p-Value von p<0,05 (Signif <1 ,3) mindestens 1,9-fach differentiell exprimiert werden, deren Expression sich um weniger als eine Zehnerpotenz unterscheidet, dh., der Quotient (Face/Breast) / (Face/CGAP) ist kleiner als 10 oder größer als 0,1. Der Vergleich der subsignifikanten Face/Breast-Daten mit unabhängigen SAGE™-Daten (Face/CGAP) bestätigt die differentielle Genexpression und validiert die Marker der Gesichtshaut.Table 10 lists all genes that are expressed differentially in facial skin (Face) compared to breast skin (Breast) with a p-value of p <0.05 (Signif <1.3) and that in facial skin (face) compared to other human tissues (except skin), whose expression profiles are represented by the CGAP database, with a p-value of p <0.05 (Signif <1, 3) at least 1.9 times are differentially expressed, the expression of which differs by less than a power of ten, that is, the quotient (face / breast) / (face / CGAP) is less than 10 or greater than 0.1. The comparison of the subsignificant face / breast data with independent SAGE ™ data (face / CGAP) confirms the differential gene expression and validates the markers of the facial skin.
In Tabelle 11 sind weitere Gene aufgelistet, die in Gesichtshaut (Face) im Vergleich zu Brusthaut (Breast) mit einem p-Value von p<0,05 (Signif <1 ,3) differentiell exprimiert werden und die in Gesichtshaut (Face) im Vergleich zu sonstigen menschlichen Geweben (außer Haut), deren Expressionsprofile durch
die CGAP-Datenbank repräsentiert werden, mit einem p-Value von p>0,05 (Signif >1,3) differentiell exprimiert werden, deren Expression sich um mehr als eine Zehnerpotenz unterscheidet, dh., der Quotient (Face/Breast) / (Face/CGAP) ist größer als 10 oder kleiner als 0,1. Der Vergleich der subsignifikanten Face/Breast- Daten mit unabhängigen SAGE™-Daten (Face/CGAP) bestätigt die differentielle Genexpression und validiert die Marker der Gesichtshaut.Table 11 lists further genes that are differentially expressed in facial skin (Face) compared to breast skin (Breast) with a p-value of p <0.05 (Signif <1, 3) and that in facial skin (Face) im Comparison to other human tissues (except skin), whose expression profiles by the CGAP database are represented, are differentially expressed with a p-value of p> 0.05 (Signif> 1.3), the expression of which differs by more than a power of ten, i.e. the quotient (face / breast) / (Face / CGAP) is greater than 10 or less than 0.1. The comparison of the subsignificant face / breast data with independent SAGE ™ data (face / CGAP) confirms the differential gene expression and validates the markers of the facial skin.
In Tabelle 12 sind weitere Gene aufgelistet, die in Gesichtshaut (Face) im Vergleich zu Brusthaut (Breast) mit einem p-Value von p<0,05 (Signif <1,3) differentiell exprimiert werden und die in Gesichtshaut (Face) im Vergleich zu sonstigen menschlichen Geweben (außer Haut), deren Expressionsprofile durch die CGAP-Datenbank repräsentiert werden, mit einem p-Value von p<0,05 (Signif <1,3) differentiell exprimiert werden, deren Expression sich um mehr als eine Zehnerpotenz unterscheidet, dh., der Quotient (Face/Breast) / (Face/CGAP) ist größer als 10 oder kleiner als 0,1.Table 12 lists further genes that are differentially expressed in facial skin (Face) compared to breast skin (Breast) with a p-value of p <0.05 (Signif <1.3) and that in facial skin (Face) im Compared to other human tissues (except skin), whose expression profiles are represented by the CGAP database, are differentially expressed with a p-value of p <0.05 (Signif <1.3), the expression of which is more than a power of ten differs, that is, the quotient (Face / Breast) / (Face / CGAP) is greater than 10 or less than 0.1.
Die zweite der vorliegenden Erfindung zugrundeliegende Aufgabe wird erfindungsgemäß gelöst durch ein Verfahren (2) zur Bestimmung der Homeostase humaner Gesichtshaut, insbesondere weiblicher Gesichtshaut, in vitro, das dadurch gekennzeichnet ist, daß man a) ein Gemisch von Proteinen, mRNA-Molekülen oder Fragmenten von Proteinen oder mRNA-Molekülen aus humaner Gesichtshaut gewinnt, b) das gewonnene Gemisch auf das Vorhandensein und gegebenenfalls die Menge von mindestens einem der Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen untersucht, die mittels Serieller Analyse der Genexpression (SAGE) als in humaner Gesichtshaut und in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut, differentiell exprimiert identifiziert werden, c) die Untersuchungsergebnisse aus b) mit den mittels Serieller Analyse der Genexpression (SAGE) identifizierten Expressionsmustern vergleicht und d) das in b) untersuchte Gemisch gesunder bzw. in Homeostase befindlicher Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in Gesichtshaut
stärker exprimiert werden als in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut, oder das in b) untersuchte Gemisch kranker bzw. in gestörter Homeostase befindlicher Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA- Molekülen enthält, die in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut stärker exprimiert werden als in Gesichtshaut.The second object underlying the present invention is achieved according to the invention by a method (2) for determining the homeostasis of human facial skin, in particular female facial skin, in vitro, which is characterized in that a) a mixture of proteins, mRNA molecules or fragments of B) the obtained mixture is examined for the presence and possibly the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which are determined by means of serial analysis of gene expression (SAGE) identified as differentially expressed in human facial skin and in other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin, c) comparing the test results from b) with the expression patterns identified by means of serial analysis of gene expression (SAGE) and d) that in b) below requested mixture of healthy or facial skin located in homeostasis if it contains predominantly proteins, mRNA molecules or fragments of proteins or mRNA molecules that are in facial skin are expressed more strongly than in other human tissues (not facial skin), especially in skin-protected areas, preferably in breast skin, or the mixture examined in b) of diseased or facial skin located in disturbed homeostasis if it predominantly assigns proteins, mRNA molecules or fragments contains proteins or mRNA molecules which are expressed more strongly in other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin than in facial skin.
Die Gewinnung des Gemisches in Schritt a) des erfindungsgemäßen Verfahrens zur Bestimmung der Homeostase humaner Gesichtshaut kann aus Vollhautproben, Hautäquivalenten, oder Zellen humaner Gesichtshaut vorgenommen werden.The mixture in step a) of the method according to the invention for determining the homeostasis of human facial skin can be obtained from whole skin samples, skin equivalents, or cells from human facial skin.
Es kann in Schritt b) des Verfahrens zur Bestimmung der Homeostase humaner Gesichtshaut ausreichend sein, das gewonnene Gemisch auf das Vorhandensein von mindestens einem der Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen zu untersuchen, die mittels Serieller Analyse der Genexpression (SAGE) als in Gesichtshaut und in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut differentiell exprimiert identifiziert werden, wenn diese ausschließlich in Gesichtshaut oder ausschließlich in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut exprimiert werden. In allen anderen Fällen muß in Schritt b) auch die Menge der differentiell exprimierten Moleküle untersucht werden, d. h., die Expression muß quantifiziert werden.In step b) of the method for determining the homeostasis of human facial 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 facial skin and in other human tissues (not facial skin), especially in skin-protected areas, preferably in breast skin, if these are exclusively in facial skin or exclusively in other human tissues (not facial skin), especially in skin-protected areas , preferably expressed in breast 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 Schritt d) des Verfahrens zur Bestimmung der Homeostase humaner Gesichtshaut wird das in b) untersuchte Gemisch gesunder humaner Gesichtshaut zugeordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in humaner Gesichtshaut stärker exprimiert werden als in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut, d. h., daß
das Gemisch entweder mehr unterschiedliche typischerweise in humaner Gesichtshaut exprimierte Verbindungen enthält, als solche, die typischerweise in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut exprimiert werden (qualitative Differenzierung), oder mehr Kopien von typischerweise in humaner Gesichtshaut exprimierten Verbindungen enthält, als typischerweise in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut vorhanden sind (quantitative Differenzierung). Für die Zuordnung zu kranker bzw. in gestörter Homeostase befindlicher humaner Gesichtshaut wird in komplementärerweise verfahren.In step d) of the method for determining the homeostasis of human facial skin, the mixture of healthy human facial skin examined in b) is assigned if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed more strongly in human facial skin than in other human tissues (not facial skin), especially in skin-protected areas, preferably in breast skin, ie that the mixture either contains more different compounds typically expressed in human facial skin than those that are typically expressed in other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin (qualitative differentiation), or more copies of typically in human Compounds expressed on facial skin contain, than are typically present in other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin (quantitative differentiation). The assignment is complementary to the assignment of the human facial skin to sick or disturbed homeostasis.
Eine bevorzugte Ausführungsform des erfindungsgemäßen Verfahrens zur Bestimmung der Homeostase humaner Gesichtshaut ist dadurch gekennzeichnet, daß man in Schritt b) das gewonnene Gemisch auf das Vorhandensein und gegebenenfalls die Menge von mindestens einem der Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen untersucht, die in den Tabellen 11 und 12 in Spalte 7 durch ihre UniGene-Accession-Number definiert werden, in Schritt c) die Untersuchungsergebnisse aus b) mit den in den Tabellen 11 und 12 in Spalte 3 und Spalte 5 angegebenen Expressionsquotienten vergleicht und in Schritt d) das in b) untersuchte Gemisch gesunder humaner Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in gesunder humaner Gesichtshaut stärker exprimiert werden als in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut, oder das in b) untersuchte Gemisch kranker bzw. in gestörter Homeostase befindlicher Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut stärker exprimiert werden als in humaner Gesichtshaut.
Eine weitere bevorzugte Ausführungsform des erfindungsgemäßen Verfahrens zur Bestimmung der Homeostase humaner Gesichtshaut ist dadurch gekennzeichnet, daß man in Schritt b) das gewonnene Gemisch auf das Vorhandensein und gegebenenfalls die Menge von mindestens einem der Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen untersucht, die in den Tabellen 9 und 10 in Spalte 7 durch ihre UniGene-Accession-Number definiert werden, in Schritt c) die Untersuchungsergebnisse aus b) mit den in den Tabellen 9 und 10 in Spalte 3 und Spalte 5 angegebenen Expressionsquotienten vergleicht und in Schritt d) das in b) untersuchte Gemisch gesunder humaner Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in gesunder humaner Gesichtshaut mindestens 1 ,9-fach so stark exprimiert werden wie in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut, oder das in b) untersuchte Gemisch kranker bzw. in gestörter Homeostase befindlicher Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut mindestens 1 ,9-fach so stark exprimiert werden wie in humaner Gesichtshaut.A preferred embodiment of the method according to the invention for determining the homeostasis of human facial 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) assigns the mixture of healthy human facial skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed more strongly in healthy human facial skin than in other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin, or that in b) examined mixture of facial skin or skin in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are preferred in other human tissues (not facial skin), in particular in skin-protected areas expressed more in breast skin than in human facial skin. A further preferred embodiment of the method according to the invention for determining the homeostasis of human facial 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 9 and 10 in column 7 by their UniGene Accession Number, in step c) compares the test results from b) with the expression quotients given in Tables 9 and 10 in column 3 and column 5 and in Step d) assigns the mixture of healthy human facial skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed in healthy human facial skin at least 1.9 times as strongly as in others human tissues (not facial skin), especially in skin-protected areas, preferably ise in breast skin, or the mixture examined in b) of diseased or facial skin in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are present in other human tissues (not facial skin), in particular expressed in skin-protected areas, preferably in breast skin, at least 1.9 times as strongly as in human facial skin.
Eine weitere bevorzugte Ausführungsform des erfindungsgemäßen Verfahrens zurAnother preferred embodiment of the inventive method for
Bestimmung der Homeostase humaner Gesichtshaut ist dadurch gekennzeichnet, daß man in Schritt b) das gewonnene Gemisch auf das Vorhandensein und gegebenenfalls die Menge von mindestens einem der Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen untersucht, die in den Tabellen 7 und 8 inDetermination of the homeostasis of human facial 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 Tables 7 and 8 in
Spalte 7 durch ihre UniGene-Accession-Number definiert werden, in Schritt c) die Untersuchungsergebnisse aus b) mit den in den Tabellen 7 und 8 in Spalte 3 und Spalte 5 angegebenen Expressionsquotienten vergleicht und in Schritt d) das in b) untersuchte Gemisch gesunder humaner Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente vonColumn 7 are defined 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) the mixture examined in b) is more healthy human facial skin if it is predominantly proteins, mRNA molecules or fragments of
Proteinen oder mRNA-Molekülen enthält, die in gesunder humaner Gesichtshaut
mindestens 3-fach so stark exprimiert werden wie in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut, oder das in b) untersuchte Gemisch kranker bzw. in gestörter Homeostase befindlicher Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut mindestens 3-fach so stark exprimiert werden wie in humaner Gesichtshaut.Contains proteins or mRNA molecules found in healthy human facial skin are expressed at least 3 times as strongly as in other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin, or the mixture of diseased facial skin or facial skin located in disturbed homeostasis, if it is predominantly proteins, Contains mRNA molecules or fragments of proteins or mRNA molecules which are expressed in other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin, at least 3 times as strongly as in human facial skin.
Eine besonders bevorzugte Ausführungsform des erfindungsgemäßen Verfahrens zur Bestimmung der Homeostase humaner Gesichtshaut ist dadurch gekennzeichnet, daß man in Schritt b) das gewonnene Gemisch auf das Vorhandensein und gegebenenfalls die Menge von mindestens einem der Proteine, mRNA-Mόleküle oder Fragmente von Proteinen oder mRNA-Molekülen untersucht, die in den Tabellen 5 und 6 in Spalte 7 durch ihre UniGene-Accession-Number definiert werden, in Schritt c) die Untersuchungsergebnisse aus b) mit den in den Tabellen 5 und 6 in Spalte 3 und Spalte 5 angegebenen Expressionsquotienten vergleicht und in Schritt d) das in b) untersuchte Gemisch gesunder humaner Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in gesunder humaner Gesichtshaut mindestens 5-fach so stark exprimiert werden wie in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut, oder das in b) untersuchte Gemisch kranker bzw. in gestörter Homeostase befindlicher Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut mindestens 5-fach so stark exprimiert werden wie in humaner Gesichtshaut.A particularly preferred embodiment of the method according to the invention for determining the homeostasis of human facial 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 5 and 6 in column 7 by their UniGene Accession Number, in step c) compares the test results from b) with the expression quotients given in Tables 5 and 6 in column 3 and column 5 and in Step d) assigns the mixture of healthy human facial 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 human facial skin as in other human tissues (not facial skin), especially in skin-protected areas, preferably e in breast skin, or the mixture examined in b) of diseased or facial skin in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are present in other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin, are expressed at least 5 times as strongly as in human facial skin.
Eine weitere besonders bevorzugte Ausführungsform des erfindungsgemäßen Verfahrens zur Bestimmung der Homeostase humaner Gesichtshaut ist dadurch gekennzeichnet, daß man
in Schritt b) das gewonnene Gemisch auf das Vorhandensein und gegebenenfalls die Menge von mindestens einem der Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen untersucht, die in Tabelle 4 in Spalte 5 durch ihre UniGene-Accession-Number definiert werden, in Schritt c) die Untersuchungsergebnisse aus b) mit den in Tabelle 5 in Spalte 3 angegebenen Expressionsquotienten vergleicht und in Schritt d) das in b) untersuchte Gemisch gesunder humaner Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in gesunder humaner Gesichtshaut mindestens 1,9-fach so stark exprimiert werden wie in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut, oder das in b) untersuchte Gemisch kranker bzw. in gestörter Homeostase befindlicher Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut mindestens 1 ,9-fach so stark exprimiert werden wie in humaner Gesichtshaut.Another particularly preferred embodiment of the method according to the invention for determining the homeostasis of human facial skin is characterized in that in step b) the mixture obtained is examined for the presence and possibly the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which are defined in Table 4 in column 5 by their UniGene accession number, in step c) compares the test results from b) with the expression quotient given in table 5 in column 3 and in step d) assigns the mixture of healthy human facial skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA Contains molecules that are expressed in healthy human facial skin at least 1.9 times as strongly as in other human tissues (not facial skin), especially in skin-protected areas, preferably in breast skin, or the mixture examined in b) of sick or in attributed to disturbed homeostasis of the facial skin if it is predominantly proteins, mRNA molecules or fragments of proteins or contains mRNA molecules which are expressed in other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin, at least 1.9 times as strongly as in human facial skin.
Eine weitere besonders bevorzugte Ausführungsform des erfindungsgemäßenAnother particularly preferred embodiment of the invention
Verfahrens zur Bestimmung der Homeostase humaner Gesichtshaut ist dadurch gekennzeichnet, daß man in Schritt b) das gewonnene Gemisch auf das Vorhandensein und gegebenenfalls die Menge von mindestens einem der Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen untersucht, die in Tabelle 3 in Spalte 5 durch ihre UniGene-Accession-Number definiert werden, in Schritt c) die Untersuchungsergebnisse aus b) mit den in Tabelle 3 in Spalte 3 angegebenen Expressionsquotienten vergleicht und in Schritt d) das in b) untersuchte Gemisch gesunder humaner Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente vonA method for determining the homeostasis of human facial 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 5 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 in step d) assigning the mixture of healthy human facial skin examined in b) if it predominantly proteins, mRNA molecules or fragments of
Proteinen oder mRNA-Molekülen enthält, die in gesunder humaner Gesichtshaut mindestens 3-fach so stark exprimiert werden wie in sonstigen menschlichenContains proteins or mRNA molecules that are expressed at least 3 times as strongly in healthy human facial skin as in other human
Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut, oder das in b) untersuchte Gemisch kranker bzw. in
gestörter Homeostase befindlicher Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut mindestens 3-fach so stark exprimiert werden wie in humaner Gesichtshaut.Tissues (not facial skin), especially in skin-protected areas, preferably in breast skin, or the mixture examined in b) of sick or in assigned to disturbed homeostasis of the facial 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 other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin become like in human facial skin.
Eine weitere besonders bevorzugte Ausführungsform des erfindungsgemäßen Verfahrens zur Bestimmung der Homeostase humaner Gesichtshaut ist dadurch gekennzeichnet, daß man in Schritt b) das gewonnene Gemisch auf das Vorhandensein und gegebenenfalls die Menge von mindestens einem der Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen untersucht, die in Tabelle 2 in Spalte 5 durch ihre UniGene-Accession-Number definiert werden, in Schritt c) die Untersuchungsergebnisse aus b) mit den in Tabelle 2 in Spalte 3 angegebenen Expressionsquotienten vergleicht und in Schritt d) das in b) untersuchte Gemisch gesunder humaner Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in gesunder humaner Gesichtshaut mindestens 5-fach so stark exprimiert werden wie in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut, oder das in b) untersuchte Gemisch kranker bzw. in gestörter Homeostase befindlicher Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut mindestens 5-fach so stark exprimiert werden wie in humaner Gesichtshaut.Another particularly preferred embodiment of the method according to the invention for determining the homeostasis of human facial 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 2 in column 5 by their UniGene Accession Number, in step c) comparing the test results from b) with the expression quotients given in table 2 in column 3 and in step d) that examined in b) Mixture of healthy human facial 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 healthy human facial skin as in other human tissues (not facial skin), in particular in skin protected areas, preferably in the breast skin, or that in b) classifies the examined mixture of diseased skin or facial skin in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are found in other human tissues (not facial skin), in particular in skin-protected areas, preferably in Breast skin are expressed at least 5 times as strongly as in human facial skin.
Eine weitere ganz besonders bevorzugte Ausführungsform des erfindungsgemäßen Verfahrens zur Bestimmung der Homeostase humaner Gesichtshaut ist dadurch gekennzeichnet, daß man in Schritt b) das gewonnene Gemisch auf das Vorhandensein und gegebenenfalls die Menge von mindestens einem der Proteine, mRNA-Moleküle oder Fragmente
von Proteinen oder mRNA-Molekülen untersucht, die in Tabelle 1 in Spalte 5 durch ihre UniGene-Accession-Number definiert werden, in Schritt c) die Untersuchungsergebnisse aus b) mit den in Tabelle 1 in Spalte 3 angegebenen Expressionsquotienten vergleicht und in Schritt d) das in b) untersuchte Gemisch gesunder humaner Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in gesunder humaner Gesichtshaut mindestens 10-fach so stark exprimiert werden wie in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut, oder das in b) untersuchte Gemisch kranker bzw. in gestörter Homeostase befindlicher Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut mindestens 10-fach so stark exprimiert werden wie in humaner Gesichtshaut.Another very particularly preferred embodiment of the method according to the invention for determining the homeostasis of human facial 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 which are defined in Table 1 in column 5 by their UniGene accession number, in step c) comparing the test results from b) with the expression quotients given in table 1 in column 3 and in step d) The mixture examined in b) is assigned to healthy human facial skin if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are expressed in healthy human facial skin at least 10 times as strongly as in other human tissues (not facial skin ), especially in skin-protected areas, preferably in breast skin, or the mixture of diseased facial skin or facial skin located in disturbed homeostasis, if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are found in others human tissues (not facial skin), especially in skin-protected areas, preferably in brus thaut are expressed at least 10 times as strongly as in human facial skin.
Man kann den Zustand der Gesichtshaut auch dadurch beschreiben, daß mehrere Marker (Expressionprodukte der für Gesichtshaut bedeutsamen Gene) quantifiziert werden, die dann untereinander in einem charakteristischen Verhältnis aktiv sein müssen, um gesunde (in Homeostase befindliche) Gesichtshaut zu repräsentieren, bzw. in einem hiervon verschiedenen charakteristischen Verhältnis aktiv sein müssen, um kranke (in gestörter Homeostase befindliche) Gesichtshaut zu repräsentieren.The condition of the facial skin can also be described by quantifying several markers (expression products of the genes which are important for facial skin), which then have to be active in a characteristic relationship to one another in order to represent healthy (in homeostasis) facial skin, or in one different characteristic ratios must be active in order to represent diseased (in disturbed homeostasis) facial skin.
Ein weiterer Gegenstand der vorliegenden Erfindung ist daher ein Verfahren (3) zur Bestimmung der Homeostase der Gesichtshaut bei Menschen, insbesondere bei Frauen, in vitro, das dadurch gekennzeichnet ist, daß man a) ein Gemisch von Proteinen, mRNA-Molekülen oder Fragmenten von Proteinen oder mRNA-Molekülen aus humaner Gesichtshaut gewinnt, b) in dem gewonnenen Gemisch mindestens zwei der Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen quantifiziert, die mittels Verfahren (1) als für Gesichtshaut bedeutsam identifiziert werden,
c) die Expressionsverhältnisse der mindestens zwei Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen zueinander bestimmt und den Expressionsquotienten bildet, d) die Expressionsverhältnisse aus c) mit den Expressionsverhältnissen vergleicht, die für die in b) quantifizierten Moleküle typischerweise in Gesichtshaut bzw. in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut vorliegen, insbesondere mit den Expressionsverhältnissen, die sich aus den Tabellen 1 bis 4, Spalte 3 bzw. den Tabellen 5 bis 12, Spalten 3 und 5 ergeben, und e) das in a) gewonnene Gemisch gesunder (in Homeostase befindlicher) humaner Gesichtshaut zuordnet, wenn die Expressionsverhältnisse der untersuchten Haut den Expressionsverhältnissen in humaner Gesichtshaut entsprechen, oder das in a) gewonnene Gemisch kranker (in gestörter Homeostase befindlicher) Gesichtshaut zuordnet, wenn die Expressionsverhältnisse der untersuchten Haut den Expressionsverhältnissen in sonstigen menschlichen Geweben (nicht Gesichtshaut), insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut entsprechen.The present invention therefore furthermore relates to a method (3) for determining the homeostasis of the facial skin in humans, 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 facial 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 facial skin by method (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 and the expression quotient is formed, d) the expression ratios from c) are compared with the expression ratios which are typically found in facial skin for the molecules quantified in b) or in other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin, in particular with the expression ratios that can be found in Tables 1 to 4, Column 3 and Tables 5 to 12, Columns 3 and 5 and e) assigns the mixture obtained in a) to healthy human facial skin (located in homeostasis) if the expression ratios of the examined skin correspond to the expression ratios in human facial skin, or assigns the mixture obtained in a) to diseased facial skin (in disturbed homeostasis) if the expression ratios of the examined skin correspond to the expression conditions in other human tissues (not facial skin), in particular in skin-protected areas, preferably in breast skin.
Vorzugsweise gewinnt man in Schritt a) der erfindungsgemäßen Verfahren zur Bestimmung der Homeostase humaner Gesichtshaut das Gemisch aus einer Hautprobe, insbesondere aus einer Vollhautprobe oder aus einer Epidermisprobe. Hierbei eröffnet die Vollhautprobe umfassendere Vergleichsmöglichkeiten mit den gleichfalls aus Vollhaut gewonnenen SAGE-Libraries. Die Epidermisprobe ist hingegen leichter zu gewinnen, beispielsweise durch Aufbringen eines Klebebandes auf die Haut und Abreißen desselben, wie in der WO 00/10579 beschrieben, auf die hiermit in vollem Umfang Bezug genommen wird.In step a) of the method according to the invention for determining the homeostasis of human facial skin, 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 einer weiteren Ausführungsform der erfindungsgemäßen Verfahren zur Bestimmung der Homeostase humaner Gesichtshaut gewinnt man in Schritt a) das Gemisch mittels Mikrodialyse. Die Technik der Mikrodialyse wird beispielsweise 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; sowie in „Cutaneous microdialysis for human in vivo dermal
absorption studies", Anderson, C. et al. ; Drugs Pharm. Sei., 1998, 91, 231-244; und auch im Internet unter http://www.microdialysis.se/techniqu.htm beschrieben, worauf hiermit in vollem Umfang Bezug genommen wird.In a further embodiment of the method according to the invention for determining the homeostasis of human facial 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. Sei., 1998, 91, 231-244; and also on the Internet at http://www.microdialysis.se/techniqu.htm, which is hereby fully described Scope is referred to.
Bei der Anwendung der Mikrodialyse führt man typischerweise eine Sonde in die Haut ein und beginnt mit einer geeigneten Trägerlösung die Sonde langsam zu spülen. Nach dem Abklingen der akuten Reaktionen nach dem Einstich liefert die Mikrodialyse Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen, die im extrazellulären Raum vorkommen und die, beispielsweise durch Fraktionierung der Trägerflüssigkeit, dann in vitro isoliert und analysiert werden können. Die Mikrodialyse ist weniger invasiv, als die Entnahme einer Vollhautprobe; sie ist aber nachteiligerweise auf die Gewinnung im extrazelulären Raum vorkommender Verbindungen beschränkt.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.
Eine weitere bevorzugte Ausführungsform der erfindungsgemäßen Verfahren zur Bestimmung der Homeostase humaner Gesichtshaut ist dadurch gekennzeichnet, daß man in Schritt b) in Verfahren (2) die Untersuchung auf das Vorhandensein und gegebenenfalls die Menge von mindestens einem der Proteine oder Proteinfragmente; bzw. in Verfahren (3) die Quantifizierung mindestens zweier Proteine oder Proteinfragmente, mittels einer Methode durchführt, die ausgewählt ist unterA further preferred embodiment of the method according to the invention for determining the homeostasis of human facial 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
Ein- oder zweidimensionaler Gelelektrophorese Affinitätschromatographie Protein-Protein-Komplexierung in Lösung iv. Massenspektrometrie, insbesondere Matrix Assistierter Laser DesorptionsOne- or two-dimensional gel electrophoresis Affinity chromatography Protein-protein complexation in solution iv. Mass spectrometry, especially matrix assisted laser desorption
Ionisation (MALDI) und insbesondere v. Einsatz von Proteinchips, oder mittels geeigneter Kombinationen dieser Methoden.Ionization (MALDI) and especially v. Use of protein chips, or by means of suitable combinations of these methods.
Diese erfindungsgemäß einsetzbaren Methoden sind in dem Übersichtsartikel von Akhilesh Pandey und Matthias Mann: „Proteomics to study genes and genomes", Nature, Volume 405, Number 6788, 837 - 846 (2000), und den dort angegebenen Referenzen beschrieben, worauf hiermit in vollem Umfang Bezug genommen wird.
Die 2D-Gelelektrophorese, wird beispielsweise in L.D. Adams, Two-dimensional Gel Electrophoresis using the Isodalt System oder in L.D. Adams & S.R. Gallagher, Two-dimensional Gel Electrophoresis using the O'Farrell System; beide in Current Protocols in Molecular Biology (1997, Eds. F.M. Ausubel et al.), Unit 10.3.1 - 10.4.13; oder in 2-D Electrophoresis-Manual; T. Berkelman, T. Senstedt; Amersham Pharmacia Biotech, 1998 (Bestell-Nr. 80-6429-60), beschrieben.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 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 electrophoresis manual; T. Berkelman, T. Senstedt; Amersham Pharmacia Biotech, 1998 (order no. 80-6429-60).
Die massenspektrometrische Charakterisierung der Proteine oder Proteinfragmente erfolgt in der Fachwelt bekannter Weise, beispielsweise wie in den folgenden Literaturstellen beschrieben: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: A. J. Link; Humana Press; Totowa; New Jersey. Darin insbesondere: Courchesne, P. L. und Patterson, S. D.; S. 487-512.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. und 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.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.
Eine weitere bevorzugte Ausführungsform der erfindungsgemäßen Verfahren zur Bestimmung der Homeostase humaner Gesichtshaut ist dadurch gekennzeichnet, daß man in Schritt b) in Verfahren (2) die Untersuchung auf das Vorhandensein und gegebenenfalls die Menge von mindestens einem der mRNA-Moleküle oder mRNA-Molekülfragmente; bzw. in Verfahren (3) die Quantifizierung mindestens zweier mRNA-Moleküle oder mRNA-Molekülfragmente mittels einer Methode durchführt, die ausgewählt ist unter Northern Blots,A further preferred embodiment of the method according to the invention for determining the homeostasis of human facial 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) 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 Transkriptase Polymerasekettenreaktion (RT-PCR), RNase-Schutzexperimente, iv. Dot-Blots, v. cDNA-Sequenzierung, vi. Klon-Hybridisierung, vii. Differential Display, viii. Subtraktive Hybridisierung,
ix. cDNA-Fragment-Fingerprinting, x. Total Gene Expression Analysis (TOGA), xi. Serielle Analyse der Genexpression (SAGE), xii. Massively Parallel Signature Sequencing (MPSS®) und insbesondere xiii. Einsatz von Nukleinsäurechips, oder mittels geeigneter Kombinationen dieser Methoden.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.
Diese erfindungsgemäß einsetzbaren Methoden sind in den Übersichtsartikeln von Akhilesh Pandey und Matthias Mann: „Proteomics to study genes and genomes", Nature, Volume 405, Number 6788, 837 - 846 (2000), und „Genomics, gene expression and DNA arrays", Nature, Volume 405, Number 6788, 827 - 836 (2000), und den dort angegebenen Referenzen beschrieben, worauf hiermit in vollem Umfang Bezug genommen wird.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 therein, to which reference is hereby made in full.
Das TOGA-Verfahren ist 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)" beschrieben, worauf hiermit vollumfänglich Bezug genommen wird.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.
Das MPSS®-Verfahren ist in der US-A-6,013,445 beschrieben, worauf hiermit in vollem Umfang Bezug genommen wird.The MPSS ® process is US Patent No. 6,013,445 described in, which reference is hereby made in its entirety.
Es können jedoch erfindungsgemäß auch andere dem Fachmann bekannte Methoden zur Untersuchung auf das Vorhandensein und gegebenenfalls die Menge von mindestens einem der Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen eingesetzt werden.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.
Eine weitere bevorzugte Ausführungsform der erfindungsgemäßen Verfahren zur Bestimmung der Homeostase humaner Gesichtshaut ist dadurch gekennzeichnet, daß man in Schritt b) auf das Vorhandensein und gegebenenfalls die Menge von 1 bis etwa 5000, bevorzugt 1 bis etwa 1000, insbesondere etwa 10 bis etwa 500, vorzugsweise etwa 10 bis etwa 250, besonders bevorzugt etwa 10 bis etwa 100 und ganz besonders bevorzugt etwa 10 bis etwa 50 der Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen untersucht, die in den
Tabellen 1 bis 4 in Spalte 5 und in den Tabellen 5 bis 12 in Spalte 7 durch ihre UniGene-Accession-Number definiert werden.A further preferred embodiment of the method according to the invention for determining the homeostasis of human facial 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 which are examined in the Tables 1 to 4 in column 5 and Tables 5 to 12 in column 7 are defined by their UniGene Accession Number.
Ein weiterer Gegenstand der vorliegenden Erfindung ist ein Test-Kit zur Bestimmung der Homeostase der Gesichtshaut bei Menschen in vitro, umfassend Mittel zur Durchführung der erfindungsgemäßen Verfahren zur Bestimmung der Homeostase humaner Gesichtshaut.Another object of the present invention is a test kit for determining the homeostasis of facial skin in humans in vitro, comprising means for carrying out the method according to the invention for determining the homeostasis of human facial skin.
Ein weiterer Gegenstand der vorliegenden Erfindung ist ein Biochip zur Bestimmung der Homeostase der Gesichtshaut bei Menschen in vitro, umfassend i. einen festen, d. h. starren oder flexiblen Träger und ii. auf diesem immobilisierte Sonden, die zur spezifischen Bindung an mindestens eines der Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen befähigt sind, die in den Tabellen 1 bis 4 in Spalte 5 und in den Tabellen 5 bis 12 in Spalte 7 durch ihre UniGene- Accession-Number definiert werden, insbesondere solche, die in den Tabellen 1 bis 4 in Spalte 5 durch folgende UniGene-Accession-Numbers definiert werden:Another object of the present invention is a biochip for determining the homeostasis of the facial 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, in Tables 1 to 4 in column 5 and in Tables 5 to 12 in column 7 their UniGene Accession Number are defined, especially those defined in Tables 1 to 4 in Column 5 by the following UniGene Accession Numbers:
• Tabelle 1 : Hs.112457, Hs.83190, Hs.80342, Hs.198862, Hs.295726, Hs.100000, Hs.334309, Hs.790, Hs.3416, Hs.277543, Hs.251531, Hs.79732, Hs.75777, Hs.99853, Hs.172928, Hs.159263;• Table 1: Hs.112457, Hs.83190, Hs.80342, Hs.198862, Hs.295726, Hs.100000, Hs.334309, Hs.790, Hs.3416, Hs.277543, Hs.251531, Hs. 79732, Hs.75777, Hs.99853, Hs.172928, Hs.159263;
• Tabelle 2: Hs.344027, Hs.245188, Hs.77910, Hs.77060, Hs.75318, Hs.74304, Hs.3416, Hs.18420, Hs.334305, Hs.287820, Hs.117938;Table 2: Hs.344027, Hs.245188, Hs.77910, Hs.77060, Hs.75318, Hs.74304, Hs.3416, Hs.18420, Hs.334305, Hs.287820, Hs.117938;
• Tabelle 3: Hs.344027, Hs.296049, Hs.2785, Hs.75445, Hs.75736, Hs.74471 , Hs.11050, Hs.334822, Hs.38991 , Hs.288998, Hs.239189, Hs.149609, Hs.17409;• Table 3: Hs.344027, Hs.296049, Hs.2785, Hs.75445, Hs.75736, Hs.74471, Hs.11050, Hs.334822, Hs.38991, Hs.288998, Hs.239189, Hs. 149609, ms. 17409;
• Tabelle 4: Hs.153179, Hs.73995, Hs.111301, Hs.119301, Hs.172928, Hs.14376.• Table 4: ms.153179, ms.73995, ms.111301, ms.119301, ms.172928, ms.14376.
Bei einem BioChip handelt es sich um ein miniaturisiertes Funktionselement mit auf einer Oberfläche immobilisierten Molekülen, insbesondere Biomolekülen, die als spezifische Interaktionspartner dienen können.
Häufig weist die Struktur dieser Funktionselemente Reihen und Spalten auf; man spricht dann von Chip-"Arrays". Da tausende von biologischen bzw. biochemischen Funktionselementen auf einem Chip angeordnet sein können, müssen diese in der Regel mit mikrotechnischen Methoden angefertigt werden. Als biologische und biochemische Funktionselemente kommen insbesondere in Frage: DNA, RNA, PNA, (bei Nukleinsäuren und ihren chemischen Derivaten können z. B. Einzelstränge, Triplex-Strukturen oder Kombinationen hiervon vorliegen), Saccharide, Peptide, Proteine (z. B. Antikörper, Antigene, Rezeptoren) und Derivate der kombinatorischen Chemie (z. B. organische Moleküle). Im allgemeinen haben BioChips eine 2D-Basisfläche für das Beschichten mit biologisch oder biochemisch funktioneilen Materialien. Die Basisflächen können beispielweise auch von Wänden einer oder mehrerer Kapillaren oder von Kanälen gebildet sein.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.
Zum Stand der Technik kann z. B. auf folgende Publikationen hingewiesen werden: Nature Genetics, Vol. 21 , Supplement (Gesamt), Jan. 1999 (BioChips); Nature Biotechnology, Vol. 16, S. 981-983, Okt. 1998 (BioChips); Trends in Biotechnology, Vol. 16, S. 301-306, Jul. 1998 (BioChips) sowie die bereits genannten Übersichtsartikel von Akhilesh Pandey und Matthias Mann: „Proteomics to study genes and genomes", Nature, Volume 405, Number 6788, 837 - 846 (2000), und „Genomics, gene expression and DNA arrays", Nature, Volume 405, Number 6788, 827 - 836 (2000), und die dort angegebenen Referenzen, worauf hiermit in vollem Umfang Bezug genommen wird.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 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.
Eine übersichtliche Darstellung der praktischen Anwendungsverfahren der DNA- Chiptechnologie liefern die Bücher „DNA Microarrays: A Practical Approach" (Editor: Mark Schena, 1999, Oxford University Press) und „Microarray Biochip Technology" (Editor: Mark Schena, 2000, Eaton Publishing), auf die hiermit in vollem Umfang Bezug genommen wird.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.
Die im Rahmen der vorliegenden Erfindung besonders bevorzugte DNA- Chiptechnologie beruht auf der Fähigkeit von Nukleinsäuren komplementäre Basenpaarungen einzugehen. Dieses als Hybridisierung bezeichnete technische Prinzip wird bereits seit Jahren bei der Southern-Blot- und Northern-Blot-Analyse
eingesetzt. Im Vergleich zu diesen herkömmlichen Methoden, bei denen lediglich einige wenige Gene analysiert werden, gestattet es die DNA-Chiptechnologie einige hundert bis zu mehreren zehntausend Genen parallel zu untersuchen. Ein DNA-Chip besteht im wesentlichen aus einem Trägermaterial (z.B. Glas oder Kunststoff), auf dem einzelsträngige, genspezifische Sonden in hoher Dichte an einer definierten Stelle (Spot) immobilisiert werden. Als problematisch wird dabei die Technik der Sonden-Applikation und die Chemie der Sonden-Immobilisierung eingeschätzt.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 used. 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 (eg 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.
Nach dem derzeitigen Stand der Technik sind mehrere Wege der Sonden- Immobilisierung realisiert:According to the current state of the art, several ways of immobilizing probes are realized:
E.M. Southern (E.M. Southern et al. (1992), Nucleic Acid Research 20, 1679-1684 und E.M. Southern et al. ( 1997), Nucleic Acid Research 25, 1155-1161) beschreibt die Herstellung von Oligonukleotidanordnungen durch direkte Synthese an einer Glasoberfläche, die mit 3-Glycidoxypropyltrimethoxysilan und anschließend mit einem Glycol derivatisiert wurde.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.
Ein ähnliches Verfahren realisiert die in situ Synthese von Oligonukleotiden mittels einer photosensitiven, kombinatorischen Chemie, die mit photolithographischen Techniken verglichen werden kann (Pease, A.C. et al. (1994), Proc. NatI Acad Sei USA 91, 5022-5026).A similar process realizes the in situ synthesis of oligonucleotides using a photosensitive, combinatorial chemistry that can be compared with photolithographic techniques (Pease, A.C. et al. (1994), Proc. NatI Acad Sei USA 91, 5022-5026).
Neben diesen auf der in s/ϊu-Synthese von Oligonukleotiden beruhenden Techniken können ebenso bereits vorhandene DNA-Moleküle an Oberflächen von Trägermaterial gebunden werden.In addition to these techniques based on s / ϊu 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) beschreibt die Immobilisierung von DNA an mit Polylysin beschichteten Glasoberflächen. Die Veröffentlichung von L.M. Smith (Guo, Z. et al. (1994), Nucleic Acid Research 22, 5456-5465) legt ein ähnliches Verfahren offen: Oligonukleotide, die eine 5'terminale Aminogruppe tragen, können an eine Glasoberfläche gebunden werden, die mit 3-Aminopropyltrimethoxysilan und anschließend mit 1,4-Phenyl- diisothioeyanat behandelt wurde.
Die Applikation der DNA-Sonden auf einem Träger kann mit einem sogenannten „Pin-Spotter" erfolgen. Dazu tauchen dünne Metallnadeln mit z.B. einem Durchmesser von 250 μm, in Sondenlösungen ein und überführen anschließend das anhängende Probenmaterial mit definierten Volumina auf das Trägermaterial des DNA-Chips.PO Brown (DeRisi et al. (1997), Science 278, 680-686) describes the immobilization of DNA on glass surfaces coated with polylysine. 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.
Bevorzugterweise erfolgt die Sondenapplikation jedoch mittels eines piezogesteuerten Nanodispensers, der ähnlich einem Tintenstrahldrucker, Sondenlösungen mit einem Volumen von 100 Picolitern kontaktfrei auf die Oberfläche des Trägermaterials aufbringt.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.
Die Immobilisierung der Sonden erfolgt z.B. wie in der EP-A-0 965 647 beschrieben: Die Generierung von DNA-Sonden erfolgt hierbei mittels PCR unter Verwendung eines sequenzspezifischen Primerpaares, wobei ein Primer am 5'- Ende modifiziert ist und einen Linker mit einer freien Aminogruppe trägt. Damit ist sichergestellt, dass ein definierter Strang der PCR-Produkte an einer Glasoberfläche gebunden werden kann, welche mit 3-Aminopropyltrimethoxysilan und anschließend mit 1 ,4-Phenyldiisothiocyanat behandelt wurde. Die genspezifischen PCR-Produkte sollen idealerweise eine definierte Nukleinsäuresequenz in einer Länge von 200-400 bp haben und nicht redundante Sequenzen beinhalten. Nach der Immobilisierung der PCR-Produkte über den derivatisierten Primer wird der Gegenstrang des PCR-Produkts durch eine Inkubation bei 96°C für 10 Min entfernt.The probes are immobilized e.g. as described in EP-A-0 965 647: The generation of DNA probes takes place here by means of PCR using a sequence-specific primer pair, a primer being modified at the 5 'end and carrying a linker with a free amino group. 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 einer für DNA-Chips typischen Anwendung wird mRNA aus zwei zu vergleichenden Zellpopulationen isoliert. Die isolierten mRNAs werden mittels reverser Transkription unter Verwendung von z.B. fluoreszenzmarkierten Nukleotiden in cDNA umgewandelt. Dabei werden die zu vergleichenden Proben mit z.B. rot bzw. grün fluoreszierenden Nukleotiden markiert. Die cDNAs werden dann mit den auf dem DNA-Chip immobilisierten Gensonden hybridisiert und anschließend die gebundenen Fluoreszenzen quantifiziert.In a typical application for DNA chips, mRNA is isolated from two cell populations to be compared. The isolated mRNAs are reverse transcribed using e.g. fluorescence-labeled nucleotides converted into cDNA. The samples to be compared are e.g. marked with 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.
Für die Herstellung kleiner (bis etwa 500 Sonden umfassender) Biochips sind die
in der DE-A-100 28 257.1-52 und in der DE-A-101 02 063.5-52 genannten Analysechips ganz besonders bevorzugt. Diese Analysechips weisen eine elektrisch adressierbare Struktur auf, die eine Elektrofokussierung der Proben gestattet. Hierduch wird es vorteilhafterweise ermöglicht, Proben unabhängig von ihrer Viskosität mit Hilfe von Elektroden an definierten Punkten eines Punktrasters (Arrays) zu fokussieren und zu immobilisieren. Durch die Fokussierfähigkeit erfolgt gleichzeitig eine Erhöhung der lokalen Konzentration der Proben und so eine höhere Spezifität. Während der Analyse selbst besteht die Möglichkeit das Testgut an die einzelnen Positionen des Arrays zu adressieren. So kann potentiell jede untersuchte Information mit der höchst möglichen Sensitivität aufgespürt werden. Eine Kreuzkontamination durch benachbarte Spots ist nahezu ausgeschlossen.They are ideal for the production of small (up to 500 probes) biochips analysis chips mentioned in DE-A-100 28 257.1-52 and in DE-A-101 02 063.5-52 are particularly preferred. 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.
Der erfindungsgemäße Biochip umfasst bevorzugt 1 bis etwa 5000, bevorzugtermaßen 1 bis etwa 1000, insbesondere etwa 10 bis etwa 500, vorzugsweise etwa 10 bis etwa 250, besonders bevorzugt etwa 10 bis etwa 100 und ganz besonders bevorzugt etwa 10 bis etwa 50 voneinander verschiedene Sonden. Die voneinander verschiedenen Sonden können jeweils in mehrfacher Kopie auf dem Chip vorhanden sein.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.
Der erfindungsgemäße Biochip umfasst bevorzugt Nukleinsäuresonden, insbesondere RNA- oder PNA-Sonden, besonders bevorzugt DNA-Sonden. Die Nukleinsäuresonden weisen bevorzugt eine Länge von etwa 10 bis etwa 1000, insbesondere etwa 10 bis etwa 800, vorzugsweise etwa 100 bis etwa 600, besonders bevorzugt etwa 200 bis etwa 400 Nukleotiden auf.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 einer weiteren bevorzugten Form umfasst der erfindungsgemäße Biochip Peptid- oder Proteinsonden, insbesondere Antikörper.In a further preferred form, the biochip according to the invention comprises peptide or protein probes, in particular antibodies.
Ein weiterer Gegenstand der vorliegenden Erfindung ist die Verwendung der Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen, die in den Tabellen 1 bis 4 in Spalte 5 und in den Tabellen 5 bis 12 in Spalte 7 durch ihre UniGene-Accession-Number definiert werden, als Marker der Gesichtshaut bei Menschen.
Ein weiterer Gegenstand der vorliegenden Erfindung ist ein Testverfahren zum Nachweis der Wirksamkeit von kosmetischen oder pharmazeutischen Wirkstoffen gegen Erkrankungen oder Beeinträchtigungen der Homeostase humaner Gesichtshaut in vitro, dadurch gekennzeichnet, daß man a) den Hautstatus humaner Gesichtshaut durch ein erfindungsgemäßes Verfahren zur Bestimmung der Homeostase humaner Gesichtshaut, oder mittels eines erfindungsgemäßen Test-Kits zur Bestimmung der Homeostase humaner Gesichtshaut, oder mittels eines erfindungsgemäßen Biochips bestimmt, b) einen Wirkstoff gegen Erkrankungen oder Beeinträchtigungen der Homeostase humaner Gesichtshaut einmal oder mehrmals auf die Gesichtshaut aufbringt, c) erneut den Hautstatus humaner Gesichtshaut durch ein erfindungsgemäßes Verfahren zur Bestimmung der Homeostase humaner Gesichtshaut, oder mittels eines erfindungsgemäßen Test-Kits zur Bestimmung der Homeostase humaner Gesichtshaut, oder mittels eines erfindungsgemäßen Biochips bestimmt, und d) die Wirksamkeit des Wirkstoffs durch den Vergleich der Ergebnisse aus a) und c) ermittelt.The present invention furthermore relates to the use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which are listed in Tables 1 to 4 in column 5 and in Tables 5 to 12 in column 7 by their UniGene accession. Number can be defined as markers of facial skin in humans. Another object of the present invention is a test method for the detection of the effectiveness of cosmetic or pharmaceutical active substances against diseases or impairments of the homeostasis of human facial skin in vitro, characterized in that a) the skin status of human facial skin is determined by an inventive method for determining the homeostasis of human facial skin , or by means of a test kit according to the invention for determining the homeostasis of human facial skin, or by means of a biochip according to the invention, b) applying an active ingredient against diseases or impairments of the homeostasis of human facial skin once or several times to the facial skin, c) again the skin status of human facial skin a method according to the invention for determining the homeostasis of human facial skin, or by means of a test kit according to the invention for determining the homeostasis of human facial skin, or by means of a bioc according to the invention hips determined, and d) the effectiveness of the active ingredient by comparing the results from a) and c) determined.
Das erfindungsgemäße Testverfahren kann mit Vollhautproben, Hautäquivalenten oder Zellen humaner Gesichtshaut durchgeführt werden.The test method according to the invention can be carried out with whole skin samples, skin equivalents or cells of human facial skin.
Ein weiterer Gegenstand der vorliegenden Erfindung ist ein Test-Kit zum Nachweis der Wirksamkeit von kosmetischen oder pharmazeutischen Wirkstoffen gegen Erkrankungen oder Beeinträchtigungen der Homeostase humaner Gesichtshaut, umfassend Mittel zur Durchführung des erfindungsgemäßen Testverfahrens.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 human facial skin, comprising means for carrying out the test method according to the invention.
Ein weiterer Gegenstand der vorliegenden Erfindung ist die Verwendung der Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen, die in den Tabellen 1 bis 4 in Spalte 5 und in den Tabellen 5 bis 12 in Spalte 7
durch ihre UniGene-Accession-Number definiert werden, zum Nachweis der Wirksamkeit von kosmetischen oder pharmazeutischen Wirkstoffen gegen Erkrankungen oder Beeinträchtigungen der Homeostase humaner Gesichtshaut.Another object of the present invention is the use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are in Tables 1 to 4 in column 5 and in Tables 5 to 12 in column 7 defined by their UniGene Accession Number, to demonstrate the effectiveness of cosmetic or pharmaceutical agents against diseases or impairments of the homeostasis of human facial skin.
Erkrankungen oder Beeinträchtigungen der Homeostase humaner Gesichtshaut umfassen erfindungsgemäß insbesondere Pathologische Zustände der Haut, wie Neurodermitis, Sonnenbrand, Psoriasis, Sklerodermie, Ichtyosis, atopische Dermatitis, Akne, Seborrhoe, Lupus erythematodes, Rosacea, Melanoma, Basalioma, Hautkarzinom, Hautsarkomin, Vitiligo, Acne, Fettige / trockene Gesichthaut.Diseases or impairments of the homeostasis of human facial skin include, in particular, pathological conditions of the skin, such as neurodermatitis, sunburn, psoriasis, scleroderma, ichthyosis, atopic dermatitis, acne, seborrhea, lupus erythematosus, rosacea, melanoma, basalioma, skin carcinoma, skin sarcoma, vitamine sarcoma, vitamins sarcoma, vitamins Oily / dry face skin.
Ein weiterer Gegenstand der vorliegenden Erfindung ist ein Screening-Verfahren zur Identifikation von kosmetischen oder pharmazeutischen Wirkstoffen gegen Erkrankungen oder Beeinträchtigungen der Homeostase humaner Gesichtshaut in vitro, das dadurch gekennzeichnet ist, daß man a) den Hautstatus humaner Gesichtshaut durch ein erfindungsgemäßes Verfahren zur Bestimmung der Homeostase humaner Gesichtshaut, oder mittels eines erfindungsgemäßen Test-Kits zur Bestimmung der Homeostase humaner Gesichtshaut, oder mittels eines erfindungsgemäßen Biochips bestimmt, b) einen potentiellen Wirkstoff gegen Erkrankungen oder Beeinträchtigungen der Homeostase humaner Gesichtshaut einmal oder mehrmals auf die Haut aufbringt, c) den Hautstatus humaner Gesichtshaut durch ein erfindungsgemäßes Verfahren zur Bestimmung der Homeostase humaner Gesichtshaut, oder mittels eines erfindungsgemäßen Test-Kits zur Bestimmung der Homeostase humaner Gesichtshaut, oder mittels eines erfindungsgemäßen Biochips bestimmt, und d) wirksame Wirkstoffe durch den Vergleich der Ergebnisse aus a) und c) bestimmt.The present invention furthermore relates to a screening method for identifying cosmetic or pharmaceutical active substances against diseases or impairments of the homeostasis of human facial skin in vitro, which is characterized in that a) the skin status of human facial skin is determined by an inventive method for determining homeostasis human facial skin, or by means of a test kit according to the invention for determining the homeostasis of human facial skin, or determined by means of a biochip according to the invention, b) applying a potential active substance against diseases or impairments of the homeostasis of human facial skin to the skin one or more times, c) the human skin status Facial skin using a method according to the invention for determining the homeostasis of human facial skin, or by means of a test kit according to the invention for determining the homeostasis of human facial skin, or using a bi ochips determined, and d) active ingredients determined by comparing the results from a) and c).
Ein weiterer Gegenstand der vorliegenden Erfindung ist die Verwendung der Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen,
die in den Tabellen 1 bis 4 in Spalte 5 und in den Tabellen 5 bis 12 in Spalte 7 durch ihre UniGene-Accession-Number definiert werden, zur Identifikation von kosmetischen oder pharmazeutischen Wirkstoffen gegen Erkrankungen oder Beeinträchtigungen der Homeostase humaner Gesichtshaut.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 1 to 4 in column 5 and in Tables 5 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 human facial skin.
Ein weiterer Gegenstand der vorliegenden Erfindung ist ein Verfahren zur Herstellung einer kosmetischen oder pharmazeutischen Zubereitung gegen Erkrankungen oder Beeinträchtigungen der Homeostase humaner Gesichtshaut, dadurch gekennzeichnet, daß man a) wirksame Wirkstoffe mit Hilfe des erfindungsgemäßen Screening- Verfahrens, oder der Verwendung zur Identifikation von kosmetischen oder pharmazeutischen Wirkstoffen gegen Erkrankungen oder Beeinträchtigungen der Homeostase humaner Gesichtshaut bestimmt und b) als wirksam befundene Wirkstoffe mit kosmetisch und pharmakologisch geeigneten und verträglichen Trägern vermischt.
Another object of the present invention is a method for producing a cosmetic or pharmaceutical preparation against diseases or impairments of the homeostasis of human facial skin, 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 active pharmaceutical ingredients against diseases or impairments of the homeostasis of human facial skin and b) active ingredients found to be effective mixed with cosmetically and pharmacologically suitable and compatible carriers.
Tabellen:tables:
Tabelle 1:Table 1:
Tabelle 3:Table 3:
Tabelle 4:
Table 4:
Tabelle 5:Table 5:
Tabelle 6:Table 6:
Tabelle 8:Table 8:
Tabelle 9:Table 9:
Tabelle 10:Table 10:
Tabellen:tables:
Tabelle 12:Table 12:
Claims
1. Verfahren zur Identifizierung der für die Gesichtshaut bedeutsamen Gene bei Menschen in vitro, dadurch gekennzeichnet, daß man a) ein erstes Gemisch von in humaner Gesichtshaut exprimierten, d. h. transkribierten und gegebenenfalls auch translatierten genetisch codierten Faktoren, also von Proteinen, mRNA-Molekülen oder Fragmenten von Proteinen oder mRNA-Molekülen aus humaner Gesichtshaut gewinnt, b) ein zweites Gemisch von in sonstigen menschlichen Geweben, insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut exprimierten, d. h. transkribierten und gegebenenfalls auch translatierten genetisch codierten Faktoren, also von Proteinen, mRNA-Molekülen oder Fragmenten von Proteinen oder mRNA-Molekülen aus sonstigen menschlichen Geweben, insbesondere aus Haut geschützter Areale, vorzugsweise aus Brusthaut gewinnt und c) die in a) und b) gewonnenen Gemische einer Seriellen Analyse der Genexpression unterwirft, und dadurch die Gene identifiziert, die in Gesichtshaut und sonstigen menschlichen Geweben, insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut unterschiedlich stark exprimiert werden.1. Method for identifying the genes important for facial skin in humans in vitro, characterized in that a) a first mixture of genes expressed in human facial skin, i.e. H. transcribed and possibly also translated genetically encoded factors, i.e. proteins, mRNA molecules or fragments of proteins or mRNA molecules from human facial skin, b) a second mixture of those expressed in other human tissues, in particular in skin of protected areas, preferably in breast skin , d. H. transcribed and possibly also translated genetically encoded factors, i.e. proteins, mRNA molecules or fragments of proteins or mRNA molecules from other human tissues, in particular from skin of protected areas, preferably from breast skin and c) those obtained in a) and b). Subjects mixtures to a serial analysis of gene expression, and thereby identifies the genes that are expressed to varying degrees in facial skin and other human tissues, in particular in skin of protected areas, preferably in breast skin.
2. Verfahren zur Bestimmung der Homeostase humaner Gesichtshaut bei Menschen in vitro, dadurch gekennzeichnet, daß man a) ein Gemisch von Proteinen, mRNA-Molekülen oder Fragmenten von Proteinen oder mRNA-Molekülen aus humaner Gesichtshaut gewinnt, b) das gewonnene Gemisch auf das Vorhandensein und gegebenenfalls die Menge von mindestens einem der Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen untersucht, die mittels Serieller Analyse der Genexpression als in humaner Gesichtshaut und sonstigen menschlichen Geweben, insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut differentiell exprimiert identifiziert werden, c) die Untersuchungsergebnisse aus b) mit den mittels Serieller Analyse der Genexpression identifizierten Expressionsmustern vergleicht und
d) das in b) untersuchte Gemisch gesunder bzw. in Homeostase befindlicher humaner Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA- Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in humaner Gesichtshaut stärker exprimiert werden als in sonstigen menschlichen Geweben, insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut, oder das in b) untersuchte Gemisch kranker bzw. in gestörter Homeostase befindlicher humaner Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in sonstigen menschlichen Geweben, insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut stärker exprimiert werden als in humaner Gesichtshaut.2. Method for determining the homeostasis of human facial skin in humans in vitro, characterized in that a) a mixture of proteins, mRNA molecules or fragments of proteins or mRNA molecules is obtained from human facial skin, b) the mixture obtained is checked for the presence and, if necessary, examines the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules that are identified by serial analysis of gene expression as being differentially expressed in human facial skin and other human tissues, in particular in skin of protected areas, preferably in breast skin c) compares the test results from b) with the expression patterns identified by serial analysis of gene expression and d) assigns the mixture examined in b) to healthy or homeostasis human facial skin if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are more strongly expressed in human facial skin than in other human tissues, in particular in skin protected areas, preferably in breast skin, or the mixture examined in b) is assigned to diseased human facial skin or one in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are found in other human tissues , especially in skin of protected areas, preferably in breast skin, are expressed more strongly than in human facial skin.
3. Verfahren nach Anspruch 2, dadurch gekennzeichnet, daß man a) in Schritt b) das gewonnene Gemisch auf das Vorhandensein und gegebenenfalls die Menge von mindestens einem der Proteine, mRNA- Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen untersucht, die in den Tabellen 11 und 12 in Spalte 7 durch ihre UniGene-Accession-Number definiert werden, b) in Schritt c) die Untersuchungsergebnisse aus b) mit den in den Tabellen 11 und 12 in Spalte 3 und Spalte 5 angegebenen Expressionsquotienten vergleicht und c) in Schritt d) das in b) untersuchte Gemisch gesunder humaner Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in gesunder humaner Gesichtshaut stärker exprimiert werden als in sonstigen menschlichen Geweben, insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut, oder das in b) untersuchte Gemisch kranker bzw. in gestörter Homeostase befindlicher Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in sonstigen menschlichen Geweben, insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut stärker exprimiert werden als in humaner Gesichtshaut.
3. The method according to claim 2, characterized in that a) 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 in the Tables 11 and 12 in column 7 are defined by their UniGene accession number, b) 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 c) in step d) assigns the mixture examined in b) to healthy human facial skin if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are more strongly expressed in healthy human facial skin than in other human tissues, in particular in skin of protected areas , preferably in breast skin, or the mixture examined in b) is assigned to sick facial skin or skin in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are found in other human tissues, in particular in protected skin Areas, preferably in breast skin, are more strongly expressed than in human facial skin.
4. Verfahren nach Anspruch 2, dadurch gekennzeichnet, daß man a) in Schritt b) das gewonnene Gemisch auf das Vorhandensein und gegebenenfalls die Menge von mindestens einem der Proteine, mRNA- Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen untersucht, die in den Tabellen 9 und 10 in Spalte 7 durch ihre UniGene-Accession-Number definiert werden, b) in Schritt c) die Untersuchungsergebnisse aus b) mit den in den Tabellen 9 und 10 in Spalte 3 und Spalte 5 angegebenen Expressionsquotienten vergleicht und c) in Schritt d) das in b) untersuchte Gemisch gesunder humaner Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in gesunder humaner Gesichtshaut mindestens 1 ,9-fach so stark exprimiert werden wie in sonstigen menschlichen Geweben, insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut, oder das in b) untersuchte Gemisch kranker bzw. in gestörter Homeostase befindlicher Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in sonstigen menschlichen Geweben, insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut mindestens 1 ,9-fach so stark exprimiert werden wie in humaner Gesichtshaut.4. The method according to claim 2, characterized in that a) 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 in the Tables 9 and 10 in column 7 are defined by their UniGene accession number, b) in step c) compares the test results from b) with the expression quotients given in tables 9 and 10 in column 3 and column 5 and c) in step d) assigns the mixture examined in b) to healthy human facial skin 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 healthy human facial skin as in other human skin Tissues, in particular in skin of protected areas, preferably in breast skin, or the mixture examined in b) is assigned to diseased facial skin or skin in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are present in other human tissues, especially in skin of protected areas, preferably in breast skin, are expressed at least 1.9 times as strongly as in human facial skin.
5. Verfahren nach Anspruch 2, dadurch gekennzeichnet, daß man a) in Schritt b) das gewonnene Gemisch auf das Vorhandensein und gegebenenfalls die Menge von mindestens einem der Proteine, mRNA- Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen untersucht, die in den Tabellen 7 und 8 in Spalte 7 durch ihre UniGene-Accession-Number definiert werden, b) in Schritt c) die Untersuchungsergebnisse aus b) mit den in den Tabellen 7 und 8 in Spalte 3 und Spalte 5 angegebenen Expressionsquotienten vergleicht und c) in Schritt d) das in b) untersuchte Gemisch gesunder humaner Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in gesunder
humaner Gesichtshaut mindestens 3-fach so stark exprimiert werden wie in sonstigen menschlichen Geweben, insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut, oder das in b) untersuchte Gemisch kranker bzw. in gestörter Homeostase befindlicher Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in sonstigen menschlichen Geweben, insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut mindestens 3-fach so stark exprimiert werden wie in humaner Gesichtshaut.5. The method according to claim 2, characterized in that a) 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 in the Tables 7 and 8 in column 7 are defined by their UniGene accession number, b) 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 c) in step d) assigns the mixture examined in b) to healthy human facial skin if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are found in healthy in human facial skin are expressed at least three times as strongly as in other human tissues, in particular in skin of protected areas, preferably in breast skin, or the mixture examined in b) is attributed to diseased or in disturbed homeostasis facial skin if it predominantly contains proteins, mRNA Contains molecules or fragments of proteins or mRNA molecules that are expressed at least three times as strongly in other human tissues, in particular in skin of protected areas, preferably in breast skin, as in human facial skin.
6. Verfahren nach Anspruch 2, dadurch gekennzeichnet, daß man a) in Schritt b) das gewonnene Gemisch auf das Vorhandensein und gegebenenfalls die Menge von mindestens einem der Proteine, mRNA- Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen untersucht, die in den Tabellen 5 und 6 in Spalte 7 durch ihre UniGene-Accession-Number definiert werden, b) in Schritt c) die Untersuchungsergebnisse aus b) mit den in den Tabellen 5 und 6 in Spalte 3 und Spalte 5 angegebenen Expressionsquotienten vergleicht und c) in Schritt d) das in b) untersuchte Gemisch gesunder humaner Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in gesunder humaner Gesichtshaut mindestens 5-fach so stark exprimiert werden wie in sonstigen menschlichen Geweben, insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut, oder das in b) untersuchte Gemisch kranker bzw. in gestörter Homeostase befindlicher Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in sonstigen menschlichen Geweben, insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut mindestens 5-fach so stark exprimiert werden wie in humaner Gesichtshaut.6. The method according to claim 2, characterized in that a) 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 in the Tables 5 and 6 in column 7 are defined by their UniGene accession number, b) in step c) compares the test results from b) with the expression quotients given in tables 5 and 6 in column 3 and column 5 and c) in step d) assigns the mixture examined in b) to healthy human facial skin if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are expressed at least 5 times as strongly in healthy human facial skin as in other human tissues, in particular in skin of protected areas, preferably in breast skin, or the mixture examined in b) is assigned to diseased facial skin or skin in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are found in other human tissues , especially in skin of protected areas, preferably in breast skin, are expressed at least 5 times as strongly as in human facial skin.
7. Verfahren nach Anspruch 2, dadurch gekennzeichnet, daß man a) in Schritt b) das gewonnene Gemisch auf das Vorhandensein und gegebenenfalls die Menge von mindestens einem der Proteine, mRNA-
Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen untersucht, die in Tabelle 4 in Spalte 5 durch ihre UniGene-Accession-Number definiert werden, b) in Schritt c) die Untersuchungsergebnisse aus b) mit den in Tabelle 4 in Spalte 3 angegebenen Expressionsquotienten vergleicht und c) in Schritt d) das in b) untersuchte Gemisch gesunder humaner Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in gesunder humaner Gesichtshaut mindestens 1 ,9-fach so stark exprimiert werden wie in sonstigen menschlichen Geweben, insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut, oder das in b) untersuchte Gemisch kranker bzw. in gestörter Homeostase befindlicher Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in sonstigen menschlichen Geweben, insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut mindestens 1 ,9-fach so stark exprimiert werden wie in humaner Gesichtshaut.7. The method according to claim 2, characterized in that a) in step b) the mixture obtained is tested for the presence and, if appropriate, the amount of at least one of the proteins, mRNA Molecules or fragments of proteins or mRNA molecules are examined, which are defined in Table 4 in column 5 by their UniGene accession number, b) in step c) compares the test results from b) with the expression quotients given in Table 4 in column 3 and c) in step d) assigns the mixture examined in b) to healthy human facial skin 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 healthy human facial skin As in other human tissues, in particular in skin of protected areas, preferably in breast skin, or the mixture examined in b) is assigned to diseased facial skin or skin in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules contains which are expressed at least 1.9 times as strongly in other human tissues, in particular in skin of protected areas, preferably in breast skin, as in human facial skin.
8. Verfahren nach Anspruch 2, dadurch gekennzeichnet, daß man a) in Schritt b) das gewonnene Gemisch auf das Vorhandensein und gegebenenfalls die Menge von mindestens einem der Proteine, mRNA- Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen untersucht, die in Tabelle 3 in Spalte 5 durch ihre UniGene-Accession-Number definiert werden, b) in Schritt c) die Untersuchungsergebnisse aus b) mit den in Tabelle 3 in Spalte 3 angegebenen Expressionsquotienten vergleicht und c) in Schritt d) das in b) untersuchte Gemisch gesunder humaner Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in gesunder humaner Gesichtshaut mindestens 3-fach so stark exprimiert werden wie in sonstigen menschlichen Geweben, insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut, oder das in b) untersuchte Gemisch kranker bzw. in gestörter Homeostase befindlicher Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder
mRNA-Molekülen enthält, die in sonstigen menschlichen Geweben, insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut mindestens 3-fach so stark exprimiert werden wie in humaner Gesichtshaut.8. The method according to claim 2, characterized in that a) 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 listed in Table 3 in column 5 are defined by their UniGene accession number, b) in step c) compares the test results from b) with the expression quotients given in table 3 in column 3 and c) in step d) the mixture examined in b) is healthier assigned to human facial skin if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are expressed at least three times as strongly in healthy human facial skin as in other human tissues, in particular in skin of protected areas, preferably in breast skin , or the mixture examined in b) is assigned to sick facial skin or skin in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or Contains mRNA molecules that are expressed at least three times as strongly in other human tissues, in particular in skin of protected areas, preferably in breast skin, as in human facial skin.
9. Verfahren nach Anspruch 2, dadurch gekennzeichnet, daß man a) in Schritt b) das gewonnene Gemisch auf das Vorhandensein und gegebenenfalls die Menge von mindestens einem der Proteine, mRNA- Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen untersucht, die in Tabelle 2 in Spalte 5 durch ihre UniGene-Accession-Number definiert werden, b) in Schritt c) die Untersuchungsergebnisse aus b) mit den in Tabelle 2 in Spalte 3 angegebenen Expressionsquotienten vergleicht und c) in Schritt d) das in b) untersuchte Gemisch gesunder humaner Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in gesunder humaner Gesichtshaut mindestens 5-fach so stark exprimiert werden wie in sonstigen menschlichen Geweben, insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut, oder das in b) untersuchte Gemisch kranker bzw. in gestörter Homeostase befindlicher Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in sonstigen menschlichen Geweben, insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut mindestens 5-fach so stark exprimiert werden wie in humaner Gesichtshaut.9. The method according to claim 2, characterized in that a) 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 shown in Table 2 in column 5 are defined by their UniGene accession number, b) in step c) compares the test results from b) with the expression quotients given in table 2 in column 3 and c) in step d) the mixture examined in b) is healthier assigned to human facial skin if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are expressed at least 5 times as strongly in healthy human facial skin as in other human tissues, in particular in skin of protected areas, preferably in breast skin , or the mixture examined in b) is assigned to diseased facial skin or skin in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are found in other human tissues, in particular in skin of protected areas, preferably in Breast skin is expressed at least 5 times as strongly as in human facial skin.
10.Verfahren nach Anspruch 2, dadurch gekennzeichnet, daß man a) in Schritt b) das gewonnene Gemisch auf das Vorhandensein und gegebenenfalls die Menge von mindestens einem der Proteine, mRNA- Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen untersucht, die in Tabelle 1 in Spalte 5 durch ihre UniGene-Accession-Number definiert werden, b) in Schritt c) die Untersuchungsergebnisse aus b) mit den in Tabelle 1 in Spalte 3 angegebenen Expressionsquotienten vergleicht und
c) in Schritt d) das in b) untersuchte Gemisch gesunder humaner Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in gesunder humaner Gesichtshaut mindestens 10-fach so stark exprimiert werden wie in sonstigen menschlichen Geweben, insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut, oder das in b) untersuchte Gemisch kranker bzw. in gestörter Homeostase befindlicher Gesichtshaut zuordnet, wenn es überwiegend Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen enthält, die in sonstigen menschlichen Geweben, insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut mindestens 10-fach so stark exprimiert werden wie in humaner Gesichtshaut.10.The method according to claim 2, characterized in that in a) 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 shown in Table 1 in column 5 can be defined by its UniGene accession number, b) in step c) compares the test results from b) with the expression quotients given in table 1 in column 3 and c) in step d) assigns the mixture examined in b) to healthy human facial skin if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are expressed at least 10 times as strongly in healthy human facial skin as in other human tissues, in particular in skin of protected areas, preferably in breast skin, or the mixture examined in b) is assigned to diseased or disturbed facial skin in homeostasis, if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which in other human tissues, especially in skin of protected areas, preferably in breast skin, are expressed at least 10 times as strongly as in human facial skin.
11.Verfahren zur Bestimmung der Homeostase der Gesichtshaut bei Menschen, insbesondere bei Frauen, in vitro, das dadurch gekennzeichnet ist, daß man a) ein Gemisch von Proteinen, mRNA-Molekülen oder Fragmenten von Proteinen oder mRNA-Molekülen aus humaner Gesichtshaut gewinnt, b) in dem gewonnenen Gemisch mindestens zwei der Proteine, mRNA- Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen quantifiziert, die mittels eines Verfahrens nach Anspruch 1 als für Gesichtshaut bedeutsam identifiziert werden, c) die Expressionsverhältnisse der mindestens zwei Proteine, mRNA- Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen zueinander bestimmt und den Expressionsquotienten bildet, d) die Expressionsverhältnisse aus c) mit den Expressionsverhältnissen vergleicht, die für die in b) quantifizierten Moleküle typischerweise in humaner Gesichtshaut bzw. in sonstigen menschlichen Geweben, insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut vorliegen, insbesondere mit den Expressionsverhältnissen, die sich aus den Tabellen 1 bis 4, Spalte 3 bzw. den Tabellen 5 bis 12, Spalten 3 und 5 ergeben, und e) das in a) gewonnene Gemisch gesunder bzw. in Homeostase befindlicher humaner Gesichtshaut zuordnet, wenn die Expressionsverhältnisse der untersuchten Haut den Expressionsverhältnissen in humaner Gesichtshaut entsprechen, oder das in a) gewonnene Gemisch kranker bzw. in gestörter
Homeostase befindlicher Gesichtshaut zuordnet, wenn die Expressionsverhältnisse der untersuchten Haut den Expressionsverhältnissen sonstigen menschlichen Geweben, insbesondere in Haut geschützter Areale, vorzugsweise in Brusthaut entsprechen.11. Method for determining the homeostasis of the facial skin in humans, especially in 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 human facial 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 facial skin using 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 relative to each other and form the expression quotient, d) compares the expression ratios from c) with the expression ratios that are typically found for the molecules quantified in b) in human facial skin or in other human tissues, in particular in skin of protected areas , preferably present in breast skin, in particular with the expression ratios that result from Tables 1 to 4, column 3 or Tables 5 to 12, columns 3 and 5, and e) the mixture obtained in a) is healthy or in homeostasis located in human facial skin, if the expression conditions of the examined skin correspond to the expression conditions in human facial skin, or the mixture obtained in a) of diseased or disturbed Facial skin in homeostasis is assigned if the expression conditions of the skin examined correspond to the expression conditions of other human tissues, in particular in skin of protected areas, preferably in breast skin.
12. Verfahren nach einem der Ansprüche 2 bis 10, dadurch gekennzeichnet, daß man die Untersuchung in Schritt b) auf das Vorhandensein und gegebenenfalls die Menge von mindestens einem der Proteine oder Proteinfragmente mittels einer Methode durchführt, die ausgewählt ist unter12. The method according to any one of claims 2 to 10, characterized in that the examination in step b) is carried out for the presence and, if appropriate, the amount of at least one of the proteins or protein fragments using a method selected from
Ein- oder zweidimensionaler GelelektrophoreseOne or two dimensional gel electrophoresis
AffinitätschromatographieAffinity chromatography
Protein-Protein-Komplexierung in Lösung iv. Massenspektrometrie, insbesondere Matrix Assistierter Laser DesorptionsProtein-protein complexation in solution iv. Mass spectrometry, especially matrix assisted laser desorption
Ionisation und insbesondere v. Einsatz von Proteinchips, oder mittels geeigneter Kombinationen dieser Methoden.Ionization and especially v. Use of protein chips, or using suitable combinations of these methods.
13. Verfahren nach Anspruch 11, dadurch gekennzeichnet, daß man die Quantifizierung mindestens zweier Proteine oder Proteinfragmente mittels einer Methode durchführt, die ausgewählt ist unter i. Ein- oder zweidimensionaler Gelelektrophorese ii. Affinitätschromatographie iii. Protein-Protein-Komplexierung in Lösung iv. Massenspektrometrie, insbesondere Matrix Assistierter Laser Desorptions13. The method according to claim 11, characterized in that the quantification of at least two proteins or protein fragments is carried out using 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
Ionisation und insbesondere v. Einsatz von Proteinchips, oder mittels geeigneter Kombinationen dieser Methoden.Ionization and especially v. Use of protein chips, or using suitable combinations of these methods.
14. Verfahren nach einem der Ansprüche 2 bis 10 und 12, dadurch gekennzeichnet, daß man die Untersuchung in Schritt b) auf das Vorhandensein und gegebenenfalls die Menge von mindestens einem der mRNA-Moleküle oder mRNA-Molekülfragmente mittels einer Methode durchführt, die ausgewählt ist unter
i. Northern Blots, ii. Reverse Transkriptase Polymerasekettenreaktion, iii. RNase-Schutzexperimente, iv. Dot-Blots, v. CDNA-Sequenzierung, vi. Klon-Hybridisierung, vii. Differential Display, viii. Subtraktive Hybridisierung, ix. cDNA-Fragment-Fingerprinting, x. Total Gene Expression Analysis xi. Serielle Analyse der Genexpression, xii. Massively Parallel Signature Sequencing und insbesondere xiii. Einsatz von Nukleinsäurechips, oder mittels geeigneter Kombinationen dieser Methoden.14. The method according to any one of claims 2 to 10 and 12, characterized in that the examination in step b) is carried out for the presence and, if appropriate, the amount of at least one of the mRNA molecules or mRNA molecule fragments using a method that is selected under i. Northern blots, ii. Reverse transcriptase polymerase chain reaction, iii. RNase protection experiments, iv. Dot blots, v. cDNA sequencing, vi. Clonal hybridization, vii. Differential Display, viii. Subtractive hybridization, ix. cDNA fragment fingerprinting, x. Total Gene Expression Analysis xi. Serial analysis of gene expression, xii. Massively Parallel Signature Sequencing and especially xiii. Use of nucleic acid chips, or using suitable combinations of these methods.
15. Verfahren nach Anspruch 11 oder 13, dadurch gekennzeichnet, daß man in Schritt b) die Quantifizierung mindestens zweier mRNA-Moleküle oder mRNA- Molekülfragmente mittels einer Methode durchführt, die ausgewählt ist unter i. Northern Blots, ii. Reverse Transkriptase Polymerasekettenreaktion, iii. RNase-Schutzexperimente, iv. Dot-Blots, v. CDNA-Sequenzierung, vi. Klon-Hybridisierung, vii. Differential Display, viii. Subtraktive Hybridisierung, ix. cDNA-Fragment-Fingerprinting, x. Total Gene Expression Analysis, xi. Massively Parallel Signature Sequencing, xii. Serielle Analyse der Genexpression, und insbesondere xiii. Einsatz von Nukleinsäurechips, oder mittels geeigneter Kombinationen dieser Methoden.
15. The method according to claim 11 or 13, characterized in that in step b) the quantification of at least two mRNA molecules or mRNA molecule fragments is carried out using a method selected from i. Northern blots, ii. Reverse transcriptase polymerase chain reaction, iii. RNase protection experiments, iv. Dot blots, v. cDNA sequencing, vi. Clonal hybridization, vii. Differential Display, viii. Subtractive hybridization, ix. cDNA fragment fingerprinting, x. Total Gene Expression Analysis, xi. Massively Parallel Signature Sequencing,xii. Serial analysis of gene expression, and in particular xiii. Use of nucleic acid chips, or using suitable combinations of these methods.
6. Verfahren nach einem der Ansprüche 2 bis 10, 12 und 14, dadurch gekennzeichnet, daß man in Schritt b) auf das Vorhandensein und gegebenenfalls die Menge von 1 bis etwa 5000, bevorzugt 1 bis etwa 1000, insbesondere etwa 10 bis etwa 500, vorzugsweise etwa 10 bis etwa 250, besonders bevorzugt etwa 10 bis etwa 100 und ganz besonders bevorzugt etwa 10 bis etwa 50 der Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen untersucht, die in den Tabellen 1 bis 4 in Spalte 5 und in den Tabellen 5 bis 12 in Spalte 7 durch ihre UniGene- Accession-Number definiert werden.6. The method according to any one of claims 2 to 10, 12 and 14, 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 most preferably about 10 to about 50 of the proteins, mRNA molecules or fragments of proteins or mRNA molecules examined in Tables 1 to 4 in column 5 and in tables 5 to 12 in column 7 are defined by their UniGene accession number.
17. Test-Kit zur Bestimmung der Homeostase der Gesichtshaut bei Menschen in vitro, umfassend Mittel zur Durchführung der Verfahren nach einem der Ansprüche 2 bis 16.17. Test kit for determining the homeostasis of facial skin in humans in vitro, comprising means for carrying out the method according to one of claims 2 to 16.
18. Biochip zur Bestimmung der Homeostase der Gesichtshaut bei Menschen in vitro, umfassend i. einen festen, d. h. starren oder flexiblen Träger und ii. auf diesem immobilisierte Sonden, die zur spezifischen Bindung an mindestens eines der Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen befähigt sind, die in den Tabellen 1 bis 4 in Spalte 5 und in den Tabellen 5 bis 12 in Spalte 7 durch ihre UniGene- Accession-Number definiert werden, insbesondere solche, die in den Tabellen 1 bis 4 in Spalte 5 durch folgende UniGene-Accession-Numbers definiert werden:18. Biochip for determining facial skin homeostasis in humans in vitro, comprising i. a fixed one, i.e. H. rigid or flexible support and ii. probes immobilized on this which are capable of specific binding to at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules listed in Tables 1 to 4 in column 5 and in Tables 5 to 12 in column 7 their UniGene accession numbers are defined, in particular those that are defined in tables 1 to 4 in column 5 by the following UniGene accession numbers:
• Tabelle 1: Hs.112457, Hs.83190, Hs.80342, Hs.198862, Hs.295726, Hs.100000, Hs.334309, Hs.790, Hs.3416, Hs.277543, Hs.251531 , Hs.79732, Hs.75777, Hs.99853, Hs.172928, Hs.159263;• Table 1: Hs.112457, Hs.83190, Hs.80342, Hs.198862, Hs.295726, Hs.100000, Hs.334309, Hs.790, Hs.3416, Hs.277543, Hs.251531 , Hs. 79732, Hs.75777, Hs.99853, Hs.172928, Hs.159263;
• Tabelle 2: Hs.344027, Hs.245188, Hs.77910, Hs.77060, Hs.75318, Hs.74304, Hs.3416, Hs.18420, Hs.334305, Hs.287820, Hs.117938;• Table 2: Hs.344027, Hs.245188, Hs.77910, Hs.77060, Hs.75318, Hs.74304, Hs.3416, Hs.18420, Hs.334305, Hs.287820, Hs.117938;
• Tabelle 3: Hs.344027, Hs.296049, Hs.2785, Hs.75445, Hs.75736, Hs.74471, Hs.11050, Hs.334822, Hs.38991 , Hs.288998, Hs.239189, Hs.149609, Hs.17409;
• Tabelle 4: Hs.153179, Hs.73995, Hs.111301, Hs.119301, Hs.172928, Hs.14376..• Table 3: Hs.344027, Hs.296049, Hs.2785, Hs.75445, Hs.75736, Hs.74471, Hs.11050, Hs.334822, Hs.38991 , Hs.288998, Hs.239189, Hs. 149609, Hs.17409; • Table 4: Hs.153179, Hs.73995, Hs.111301, Hs.119301, Hs.172928, Hs.14376..
19. Biochip nach Anspruch 18, umfassend 1 bis etwa 5000, bevorzugt 1 bis etwa 1000, insbesondere etwa 10 bis etwa 500, vorzugsweise etwa 10 bis etwa 250, besonders bevorzugt etwa 10 bis etwa 100 und ganz besonders bevorzugt etwa 10 bis etwa 50 voneinander verschiedene Sonden.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 most preferably about 10 to about 50 of each other different probes.
20. Biochip nach Anspruch 18 oder 19, umfassend Nukleinsäuresonden, insbesondere RNA- oder PNA-Sonden, besonders bevorzugt DNA-Sonden.20. Biochip according to claim 18 or 19, comprising nucleic acid probes, in particular RNA or PNA probes, particularly preferably DNA probes.
21. Biochip nach Anspruch 20, umfassend Sonden mit einer Länge von etwa 10 bis etwa 1000, insbesondere etwa 10 bis etwa 800, vorzugsweise etwa 100 bis etwa 600, besonders bevorzugt etwa 200 bis etwa 400 Nukleotiden.21. Biochip according to claim 20, comprising probes with a length of about 10 to about 1000, in particular about 10 to about 800, preferably about 100 to about 600, particularly preferably about 200 to about 400 nucleotides.
22. Biochip nach Anspruch 18 oder 19, umfassend Peptid- oder Proteinsonden, insbesondere Antikörper.22. Biochip according to claim 18 or 19, comprising peptide or protein probes, in particular antibodies.
23. Verwendung der Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen, die in den Tabellen 1 bis 4 in Spalte 5 und in den Tabellen 5 bis 12 in Spalte 7 durch ihre UniGene-Accession-Number definiert werden, als Marker der Gesichtshaut bei Menschen.23. Use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules defined by their UniGene accession number in Tables 1 to 4 in column 5 and in Tables 5 to 12 in column 7 as markers the facial skin in humans.
24. Testverfahren zum Nachweis der Wirksamkeit von kosmetischen oder pharmazeutischen Wirkstoffen gegen Erkrankungen oder Beeinträchtigungen der Homeostase humaner Gesichtshaut in vitro, dadurch gekennzeichnet, daß man a) den Hautstatus humaner Gesichtshaut durch ein Verfahren nach einem der - Ansprüche 2 bis 15, oder mittels eines Test-Kits nach Anspruch 17, oder mittels eines Biochips nach einem der Ansprüche 18 bis 22, bestimmt, b) einen Wirkstoff gegen Erkrankungen oder Beeinträchtigungen der Homeostase humaner Gesichtshaut einmal oder mehrmals auf die Gesichtshaut aufbringt,
c) erneut den Hautstatus humaner Gesichtshaut durch ein Verfahren nach einem der Ansprüche 2 bis 15, oder mittels eines Test-Kits nach Anspruch 17, oder mittels eines Biochips nach einem der Ansprüche 18 bis 22, bestimmt, und d) die Wirksamkeit des Wirkstoffs durch den Vergleich der Ergebnisse aus a) und c) ermittelt.24. Test method for demonstrating the effectiveness of cosmetic or pharmaceutical active ingredients against diseases or impairments of the homeostasis of human facial skin in vitro, characterized in that a) the skin status of human facial skin is determined by a method according to one of claims 2 to 15, or by means of a test -Kits according to claim 17, or determined 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 human facial skin once or several times to the facial skin, c) the skin status of human facial skin is again determined by a method according to one of claims 2 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 comparing the results from a) and c).
25. Test-Kit zum Nachweis der Wirksamkeit von kosmetischen oder pharmazeutischen Wirkstoffen gegen Erkrankungen oder Beeinträchtigungen der Homeostase humaner Gesichtshaut, umfassend Mittel zur Durchführung des Testverfahrens gemäß Anspruch 24.25. Test kit for detecting the effectiveness of cosmetic or pharmaceutical active ingredients against diseases or impairments of the homeostasis of human facial skin, comprising means for carrying out the test method according to claim 24.
26. Verwendung der Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen, die in den Tabellen 1 bis 4 in Spalte 5 und in den Tabellen 5 bis 12 in Spalte 7 durch ihre UniGene-Accession-Number definiert werden, zum Nachweis der Wirksamkeit von kosmetischen oder pharmazeutischen Wirkstoffen gegen Erkrankungen oder Beeinträchtigungen der Homeostase humaner Gesichtshaut.26. Use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules defined in Tables 1 to 4 in column 5 and in Tables 5 to 12 in column 7 by their UniGene accession number for detection the effectiveness of cosmetic or pharmaceutical active ingredients against diseases or impairments of the homeostasis of human facial skin.
27. Screening-Verfahren zur Identifikation von kosmetischen oder pharmazeutischen Wirkstoffen gegen Erkrankungen oder Beeinträchtigungen der Homeostase humaner Gesichtshaut in vitro, das dadurch gekennzeichnet ist, daß man a) den Hautstatus humaner Gesichtshaut durch ein Verfahren nach einem der Ansprüche 2 bis 15, oder mittels eines Test-Kits nach Anspruch 17, oder mittels eines Biochips nach einem der Ansprüche 18 bis 22, bestimmt, b) einen potentiellen Wirkstoff gegen Erkrankungen oder Beeinträchtigungen der Homeostase humaner Gesichtshaut einmal oder mehrmals auf die Haut aufbringt, c) den Hautstatus humaner Gesichtshaut durch ein Verfahren nach einem der Ansprüche 2 bis 15, oder mittels eines Test-Kits nach Anspruch 17, oder mittels eines Biochips nach einem der Ansprüche 18 bis 22, bestimmt,
d) wirksame Wirkstoffe durch den Vergleich der Ergebnisse aus a) und c) ermittelt.27. Screening method for identifying cosmetic or pharmaceutical active ingredients against diseases or impairments of the homeostasis of human facial skin in vitro, which is characterized in that a) the skin status of human facial skin is determined by a method according to one of claims 2 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, determined, b) a potential active ingredient against diseases or impairments of the homeostasis of human facial skin is applied to the skin once or several times, c) the skin status of human facial skin is determined by a Method according to one of claims 2 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, determined, d) effective active ingredients determined by comparing the results from a) and c).
28. Verwendung der Proteine, mRNA-Moleküle oder Fragmente von Proteinen oder mRNA-Molekülen, die in den Tabellen 1 bis 4 in Spalte 5 und in den Tabellen 5 bis 12 in Spalte 7 durch ihre UniGene-Accession-Number definiert werden, zur Identifikation von kosmetischen oder pharmazeutischen Wirkstoffen gegen Erkrankungen oder Beeinträchtigungen der Homeostase humaner Gesichtshaut.28. Use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules defined in Tables 1 to 4 in column 5 and in Tables 5 to 12 in column 7 by their UniGene accession number for identification of cosmetic or pharmaceutical active ingredients against diseases or impairments of the homeostasis of human facial skin.
29. Verfahren zur Herstellung einer kosmetischen oder pharmazeutischen Zubereitung gegen Erkrankungen oder Beeinträchtigungen der Homeostase humaner Gesichtshaut, dadurch gekennzeichnet, daß man a) wirksame Wirkstoffe mit Hilfe des Screening-Verfahrens nach Anspruch 27, oder der Verwendung nach Anspruch 28 bestimmt und b) als wirksam befundene Wirkstoffe mit kosmetisch und pharmakologisch geeigneten und verträglichen Trägern vermischt.
29. A process for producing a cosmetic or pharmaceutical preparation against diseases or impairments of the homeostasis of human facial skin, characterized in that a) active active ingredients are determined using the screening method according to claim 27, or the use according to claim 28 and b) as effective Active ingredients found to be mixed with cosmetically and pharmacologically suitable and compatible carriers.
Priority Applications (2)
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|---|---|---|---|
| EP03789223A EP1573064A2 (en) | 2002-12-20 | 2003-12-11 | Method for determining markers of human facial skin |
| AU2003293839A AU2003293839A1 (en) | 2002-12-20 | 2003-12-11 | Method for determining markers of human facial skin |
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|---|---|---|---|
| DE2002160928 DE10260928A1 (en) | 2002-12-20 | 2002-12-20 | Method for the determination of markers of human facial skin |
| DE10260928.4 | 2002-12-20 |
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| WO2004059001A2 true WO2004059001A2 (en) | 2004-07-15 |
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| PCT/EP2003/014068 WO2004059001A2 (en) | 2002-12-20 | 2003-12-11 | Method for determining markers of human facial skin |
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| EP (1) | EP1573064A2 (en) |
| AU (1) | AU2003293839A1 (en) |
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| WO2015031708A1 (en) * | 2013-08-30 | 2015-03-05 | The Procter & Gamble Company | Methods of identifying cosmetic agents for treating periorbital dyschromia and systems therefor |
| NO20170739A1 (en) * | 2017-05-04 | 2018-11-05 | Patogen As | Novel virus in Fish and Method for detection |
| NO344051B1 (en) * | 2017-05-04 | 2019-08-26 | Patogen As | Novel virus in Fish and Method for detection |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2003293839A1 (en) | 2004-07-22 |
| EP1573064A2 (en) | 2005-09-14 |
| WO2004059001A3 (en) | 2005-03-03 |
| DE10260928A1 (en) | 2004-07-08 |
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