EP1573064A2 - Procede de determination de marqueurs de la peau du visage humain - Google Patents

Procede de determination de marqueurs de la peau du visage humain

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Publication number
EP1573064A2
EP1573064A2 EP03789223A EP03789223A EP1573064A2 EP 1573064 A2 EP1573064 A2 EP 1573064A2 EP 03789223 A EP03789223 A EP 03789223A EP 03789223 A EP03789223 A EP 03789223A EP 1573064 A2 EP1573064 A2 EP 1573064A2
Authority
EP
European Patent Office
Prior art keywords
skin
proteins
mrna molecules
facial skin
fragments
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03789223A
Other languages
German (de)
English (en)
Inventor
Dirk Petersohn
Kordula Schlotmann
Thomas Gassenmeier
Olaf HOLTKÖTTER
Marcus Conradt
Kay Hofmann
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Henkel AG and Co KGaA
Original Assignee
Henkel AG and Co KGaA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Henkel AG and Co KGaA filed Critical Henkel AG and Co KGaA
Publication of EP1573064A2 publication Critical patent/EP1573064A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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.

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Abstract

La présente invention concerne un procédé de détermination in vitro de l'homéostasie de la peau du visage humain, des ensembles de test, des puces biologiques destinées à la détermination de marqueurs de la peau du visage humain, ainsi que l'utilisation de protéines, de molécules d'ARNm ou de fragments de protéines ou de molécules d'ARNm en tant que marqueurs de la peau du visage humain. L'invention concerne également un procédé de test destiné à la détection de l'efficacité d'agents cosmétiques ou pharmaceutiques destinés au traitement de la peau du visage humain, un procédé de criblage destiné à l'identification d'agents cosmétiques ou pharmaceutiques destinés au traitement de la peau du visage humain, ainsi qu'un procédé de fabrication d'une composition cosmétique ou pharmaceutique destinée au traitement de la peau du visage humain.
EP03789223A 2002-12-20 2003-12-11 Procede de determination de marqueurs de la peau du visage humain Withdrawn EP1573064A2 (fr)

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DE2002160928 DE10260928A1 (de) 2002-12-20 2002-12-20 Verfahren zur Bestimmung von Markern humaner Gesichtshaut
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PCT/EP2003/014068 WO2004059001A2 (fr) 2002-12-20 2003-12-11 Procede de determination de marqueurs de la peau du visage humain

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FR2866233B1 (fr) 2004-02-18 2008-02-08 Vincience Composition dermatologique et/ou cosmetique contenant des polypeptides
FR2948021A1 (fr) 2009-07-16 2011-01-21 Oreal Utilisation cosmetique de polypeptides de type lacritine
US20150125559A1 (en) * 2013-08-30 2015-05-07 The Procter & Gamble Company Method of Identifying Cosmetic Agents For Treating Periorbital Dyschromia and Systems Therefor
WO2015105190A1 (fr) * 2014-01-10 2015-07-16 学校法人順天堂 Méthode d'évaluation du potentiel métastatique du cancer de l'endomètre en direction des ganglions lymphatiques
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WO2004059001A2 (fr) 2004-07-15

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