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EP1573053A2 - Procede de determination de l'homeostasie de peau pileuse - Google Patents

Procede de determination de l'homeostasie de peau pileuse

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Publication number
EP1573053A2
EP1573053A2 EP03767789A EP03767789A EP1573053A2 EP 1573053 A2 EP1573053 A2 EP 1573053A2 EP 03767789 A EP03767789 A EP 03767789A EP 03767789 A EP03767789 A EP 03767789A EP 1573053 A2 EP1573053 A2 EP 1573053A2
Authority
EP
European Patent Office
Prior art keywords
skin
proteins
mrna molecules
hairy
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Withdrawn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
EP03767789A
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 EP1573053A2 publication Critical patent/EP1573053A2/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Withdrawn legal-status Critical Current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
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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 the homeostasis of hairy skin in vitro, test kits and biochips for determining markers of hairy skin and the use of proteins, mRNA molecules or fragments of proteins or mRNA molecules as markers of hairy skin; furthermore a test method for the detection of the effectiveness of cosmetic or pharmaceutical active substances for the treatment of hairy skin as well as a screening method for the identification of cosmetic or pharmaceutical active substances for the treatment of hairy skin and a method for the production of a cosmetic or pharmaceutical preparation for the treatment of hairy skin.
  • Every living cell is able to react to signals from its environment.
  • the reactions of the cells are realized by an orderly regulation of the gene expression, so that the metabolism of cells is not static but very dynamic.
  • 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 human skin has hair down to the lips, the palms of the hands and the soles of the feet.
  • the inconspicuous vellus hairs do not only differ macroscopically e.g. of the cosmetically relevant hair on the head.
  • the hair follicles but also the hair itself can be differentiated microscopically. Which biochemical and molecular biological mechanisms underlie these differences is largely unknown.
  • a relevant characteristic of hair and its follicles is that with age the cells lose their ability to maintain the organ's homeostasis. For example, the number of hair follicles per unit area decreases over time. The structure of the hair also changes, for example by reducing the hair diameter. With age, certain cells of the hair follicle often no longer produce hair pigments - the hair gray. Which molecular mechanisms underlie this development has so far been largely unclear.
  • Effective cosmetic or pharmaceutical hair products should have a positive effect on the broadest possible range of molecular processes in the hair follicle. So far, however, only a few molecular reaction mechanisms have been described in the hair follicle, which can thus serve as a target for cosmetic hair products.
  • 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.
  • 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
  • mRNA molecules are present in the cell in concentrations between a few and several hundred copies.
  • the weakly expressed genes have not been available to previous analysis techniques or have been very difficult to access, but can certainly play a decisive role in the hair follicle.
  • the transcriptome i.e. the entirety of all transcribed genes of the human hair follicle, has not been described to date.
  • Transcriptome analyzes of the skin using various methods, including SAGE TM analysis, are state of the art. However, isolated keratinocytes (in vitro) or epidermal explants are used, which - as above explained - do not represent models representative of complex skin events.
  • 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 hairy skin cannot be found in these publications.
  • the object of the present invention is to identify as large a part as possible of the genes which are important for the hair on the skin.
  • methods for determining the homeostasis of hairy skin are to be provided using the identified genes.
  • This first object is achieved according to the invention by a method (1) for
  • SAGE serial analysis of gene expression
  • hairless is also to be understood as meaning the hair with the fine and barely visible vellus hair.
  • the method according to the invention advantageously makes it possible to understand the complex process of hair formation and the causal relationships between the changes in the hair. Only with this knowledge can new concepts for cosmetic hair products be developed that have an effect on the broad spectrum of gene expression in the hair follicle.
  • the SAGE TM analysis carried out in the context of the method according to the invention shows for the first time in a very comprehensive manner which genes are expressed in (with the main hair) hairy scalp and which genes are expressed there differently than in skin with vellus hair.
  • SAGE TM serial analysis of gene expression
  • the comparison of the transcriptome of hairy skin with the transcriptome of hairless skin makes it possible to distinguish between genes that are relevant and not relevant for the hair on the skin.
  • SAGE TM analysis Human skin from healthy female donors was used for SAGE TM analysis.
  • the SAGE TM analysis was carried out as described in EP-A-0 761 822 and in Velculescu, VE et al., 1995 Science 270, 484-487.
  • This technique also allows the identification and quantification of the genes expressed in hairy scalp.
  • the comparison of the transcriptome of hairy scalp with the transcriptome of facial skin allows the identification of relevant genes for hairy scalp. These can be genes that are particularly strongly expressed in hairy scalp or also genes that are characterized in that they are only expressed slightly compared to hairless facial skin.
  • the SAGE TM analysis is a technique with special sensitivity and surprisingly shows even interindividual differences in the gene expression profile.
  • the comparison to the transcriptome of hairless skin is therefore particularly effective when the analyzed tissue is from an individual come from a donor.
  • interindividual differences in gene expression are negligible.
  • hairy tissues accumulate from a patient from the area above the ear and the area behind the ear.
  • skin tissue in front of the ear is removed that - at least in female donors - only vellus
  • the analysis of such tissue samples therefore allows the description of the transcriptome of hairy scalp and hairless (or only covered with vellus hair) facial skin with the exclusion of interindividual gene expression differences.
  • the hairy skin For hairy skin, an increased number of tags can be shown for genes that are expressed in the hair follicle or in hair appendages.
  • the hairy skin In addition to an increased level of keratin, which is typical of hair and hair follicles, the hairy skin also has, for example, more FGF7, a transcription factor involved in hair development.
  • Dermcidin and cystatin, both involved in the defense against bacterial and viral attacks in the hair shaft also show a significantly increased expression in the hairy skin.
  • the expression of differentiation-dependent genes of the interfollicular epidermis is in the hairless skin intensified, which is reflected in the results of the investigation.
  • keratin 10 At the top of the differentially expressed genes is keratin 10, a typical marker for the differentiation of stratified epithelia.
  • Keratin 1 the partner of Keratin 10, does not show these differences in expression.
  • the analysis of further differentiation-dependent expressed genes also confirms their increased expression in the hairless skin (Tab. 2).
  • another keratin, keratin 2e is mainly expressed in hairless skin, which is also observed in the analysis carried out.
  • the expression of keratins 5, 14 and 15 expressed in the basal layer of the epidermis and in the hair follicles is almost identical in both samples (Tab. 2).
  • Table 1 lists markers which, according to literature knowledge, are differentially in the hair follicle in the
  • Table 2 shows differentiation-dependent genes of the interfollicular epidermis.
  • HF hair follicle
  • ORS Outer Root Sheath (Outer Root Sheath)
  • IRS Inner Root Sheath (Inner Root Sheath)
  • Tables 3 to 12 contain a detailed list of the genes determined with the aid of the method according to the invention and differentially expressed in hairy and hairless skin, with indication
  • the quotient in column 3 indicates the strength of the differential expression, i.e. i.e. by which factor the respective gene is expressed more strongly in hairy scalp (scalp) than in hairless facial skin (face), or vice versa.
  • the quotient in column 5 indicates the strength of the differential expression, i. i.e. by which factor the respective gene is expressed more strongly in hairy scalp (scalp) than in hairless body skin (breast), or vice versa.
  • the respective genes or gene products are disclosed in the database of the National Center for Biotechnology Information (NCBI) under their UniGene Accession Number. This database is accessible on the Internet at the following address: http://www.ncbi.nlm.nih.gov/. The genes or gene products are also available at http://www.ncbi.nlm.nih.gov/UniGene/Hs.Home.html or http://www.ncbi.nlm.nih.gov/genome/ guide directly accessible.
  • Table 3 lists all genes that are at least 10-fold differentially expressed in hairy scalp compared to hairless facial skin with a p-value of p> 0.05 (Signif> 1.3).
  • Table 4 lists all genes that are expressed differentially in hairy scalp (scalp) compared to hairless facial skin (face) with a p-value of p> 0.05 (Signif> 1, 3) at least 5-fold.
  • Table 5 lists all genes that are at least 3-fold differentially expressed in hairy scalp compared to hairless facial skin with a p-value of p> 0.05 (Signif> 1, 3).
  • Table 6 lists all genes that are expressed at least 1.9 times differentially in hairy scalp compared to hairless facial skin with a p-value of p> 0.05 (Signif> 1, 3) ,
  • Table 7 lists all genes that are expressed differentially in hairy scalp (scalp) compared to hairless facial skin (face) with a p-value of p ⁇ 0.05 (Signif ⁇ 1, 3) and that in hairy scalp (scalp) compared to hairless body skin (breast) with a p-value of p> 0.05 (Signif> 1, 3) at least 5-fold differentially expressed.
  • the comparison of the subsignificant scalp / face data with independent SAGE TM experiments (scalp / breast) confirms the differential gene expression and validates the markers of the hairy skin.
  • Table 8 lists all the genes that are expressed differentially in hairy scalp (scalp) compared to hairless facial skin (face) with a p-value of p ⁇ 0.05 (Signif ⁇ 1, 3) and that in hairy scalp (scalp) compared to hairless body skin (breast) with a p- Value of p ⁇ 0.05 (Signif ⁇ 1.3) are expressed at least 5-fold differentially, the expression of which differs by less than a power of ten, ie the quotient (scalp / face) / (scalp / breast) is smaller than 10 or greater than 0.1.
  • the comparison of the subsignificant scalp / face data with independent SAGE TM experiments (scalp / breast) confirms the differential gene expression and validates the markers of the hairy skin.
  • Scalp Errr compared to hairless facial skin (Face) with a p-value of p ⁇ 0.05 (Signif ⁇ 1, 3) at least 3 times differentially expressed and that in hairy scalp (scalp) compared to hairless body skin (Breast) with a p-value of p> 0.05 (Signif> 1, 3) are expressed at least 3 times differentially.
  • the comparison of the subsignificant scalp / face data with independent SAGE TM experiments confirms the differential gene expression and validates the markers of the hairy skin.
  • Table 10 lists all genes that are at least 3-fold differentially expressed in hairy scalp compared to hairless facial skin with a p-value of p ⁇ 0.05 (Signif ⁇ 1, 3) and that in hairy scalp (scalp) compared to hairless body skin (breast) with a p-value of p ⁇ 0.05 (Signif ⁇ 1, 3) at least 3-fold differentially expressed, the expression of which differs by less than a power of ten, that is, the quotient (scalp / face) / (scalp / breast) is less than 10 or greater than 0.1.
  • the comparison of the subsignificant scalp / face data with independent SAGE TM experiments (scalp / breast) confirms the differential gene expression and validates the markers of the hairy skin.
  • Table 11 lists all genes that are expressed differentially in hairy scalp (scalp) compared to hairless facial skin (face) with a p-value of p ⁇ 0.05 (Signif ⁇ 1.3) at least 1.9-fold and which are expressed differentially in hairy scalp (scalp) compared to hairless body skin (breast) with a p-value of p> 0.05 (Signif> 1, 3) at least 1.9-fold.
  • Comparison of subsignificant scalp / face data with independent SAGE TM experiments (scalp / breast) confirm the differential gene expression and validate the markers of the hairy skin.
  • Table 12 lists all genes that are expressed at least 1.9 times differentially in hairy scalp compared to hairless facial skin with a p-value of p ⁇ 0.05 (Signif ⁇ 1, 3) and in hairy scalp (scalp) compared to hairless body skin (Breast) with a p-VäT ⁇ e of p ⁇ 0.05 (signif ⁇ 1, 3) at least T, 9-fan-shaped red differentially "exf5rirniert whose expression is less as a power of ten, ie the quotient (Scalp / Face) / (Scalp / Breast) is less than 10 or greater than 0.1.
  • the comparison of the subsignificant Scalp / Face data with independent SAGE TM experiments (Scalp / Breast) confirms the differential gene expression and validates the markers of the hairy skin.
  • the second object on which YQ - Jjßg ---- d-aD invention is based is achieved according to the invention by a method (2) for determining the homeostasis of hairy skin in humans, in particular women, in vitro, which is characterized in that a ) a mixture of proteins, mRNA molecules or fragments of proteins or mRNA molecules from hairy human skin is obtained, b) the mixture obtained for the presence and optionally the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA -Molecules examined, which are identified by means of serial analysis of gene expression (SAGE) as differentially expressed in hairy and hairless skin, c) the test results from b) are compared with the expression patterns identified by means of serial analysis of gene expression (SAGE) and d) that in b ) investigated mixture of healthy or hairy skin in homeostasis if it predominantly contains proteins , mRNA molecules or fragments of proteins or mRNA molecules that are more strongly expressed in hair
  • the mixture in step a) of the method according to the invention for determining the homeostasis of hairy skin can be obtained from whole skin samples, hairy skin equivalents, isolated hair follicles, hair follicle equivalents or cells of hairy skin.
  • step b) of the method for determining the homeostasis of hairy skin it may be sufficient to examine the mixture obtained for the presence of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which can be determined by means of serial analysis of the gene expression ( SAGE) can be identified as differentially expressed in hairy and hairless skin if they are expressed exclusively in hairy or hairless skin only. In all other cases, the amount of differentially expressed molecules must also be examined in step b). that is, expression must be quantified.
  • SAGE serial analysis of the gene expression
  • step d) of the method for determining the homeostasis of hairy skin the mixture examined in b) of healthy hairy skin is assigned if it is predominantly proteins, mRNA molecules or fragments of proteins or Contains mRNA molecules that are more strongly expressed in hairy skin than in hairless skin, ie that the mixture contains either more different compounds typically expressed in hairy skin than those that are typically expressed in hairless skin (qualitative differentiation), or more Contains copies of compounds typically expressed in hairy skin than are typically present in hairless skin (quantitative differentiation).
  • the assignment to diseased or " 1rr ⁇ e ⁇ oTteT ⁇ Hömeö ⁇ stäase hairy skin is followed in a complementary manner.
  • a preferred embodiment of the method according to the invention for determining the homeostasis of hairy skin is characterized in that in step b) the mixture obtained is examined for the presence and optionally the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules , which are defined in Tables 11 and 12 in column 7 by their UniGene Accession Number, in step c) compares the test results from b) with the expression quotients given in Tables 11 and 12 in column 3 and column 5 and in step d) the mixture examined in b) is assigned to healthy hairy skin if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed in healthy hairy skin at least 1.9 times as strongly as in hairless skin , or assigns the mixture examined in b) to diseased or hairy skin in disturbed homeostasis, if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are expressed at least 1.9 times as strongly in hairless skin as in hairy skin.
  • step b) Determination of the homeostasis of hairy skin is characterized in that in step b) the mixture obtained is checked for the presence and, if appropriate, the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are defined in Tables 9 and 10 in column 7 by their UniGene Accession Number, in step c) the test results from b) with those in Tables 9 and 10 in column 3 and column 5 given expression quotient and in step d) assigns the mixture of healthy hairy skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are at least 3 times in healthy hairy skin barren "the ih ⁇ b) l tersücl ⁇ fe mixture diseased or disturbed homeostasis in-hairy skin allocates when it contains mainly proteins, mRNA molecules or fragments of proteins or mRNA molecules that fold in hairless skin at least 3 so highly expressed become like in hairy skin.
  • a further preferred embodiment of the method according to the invention for determining the homeostasis of hairy skin is characterized in that in step b) the mixture obtained is checked for the presence and optionally the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules examined, which are defined in Tables 7 and 8 in column 7 by their UniGene Accession Number, in step c) compares the test results from b) with the expression quotients given in Tables 7 and 8 in column 3 and column 5 and in Step d) assigns the mixture of healthy hairy skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least 5 times as strongly in healthy hairy skin as in hairless skin, or assigns the mixture examined in b) to diseased or hairy skin in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least 5 times as strongly in hairless skin as in hairy skin.
  • a particularly preferred embodiment of the method according to the invention for determining the homeostasis of hairy skin is characterized in that in step b) the mixture obtained is checked for the presence and optionally the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules examined, which are defined in Table 6 in column 7 by their UniGene Accession Number, in step c) the ⁇ Ontersucl ⁇ üTrgsel ⁇ e ⁇ ⁇ is ⁇ eä ⁇ sb) ⁇ rnit with " deh " in table 6 ⁇ in " S ' column 3 and column 5 expressed expression quotient and in step d) assigns the mixture of healthy hairy skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are at least 1.9 times as good in healthy hairy skin are strongly expressed as in hairless skin, or the mixture examined in b) of sick or hairy in disturbed homeostasis contains proteins or mRNA molecules that are expressed at
  • the method for determining the homeostasis of hairy skin is characterized in that in step b) the mixture obtained is examined for the presence and, if appropriate, the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which are shown in Table 5 defined in column 7 by their UniGene accession number, in step c) comparing the test results from b) with the expression quotients given in table 5 in column 3 and column 5 and in step d) the mixture of healthy hairy skin examined in b) assigned if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least 3 times as strongly in healthy hairy skin as in hairless skin, or which was examined in b)
  • a further particularly preferred embodiment of the method for determining the homeostasis of the scalp is characterized in that in step b) the mixture obtained in the presence and optionally iie _ M "ertge ⁇ ⁇ n _ min" de "ste ⁇ s ⁇ a _ of the proteins ; mRNA molecules or fragments of proteins or mRNA molecules examined, which are defined in Table 4 in column 7 by their UniGene Accession Number, in step c) the test results from b) with those in Table 4 in column 3 and column 5 indicated expression quotient and in step d) assigns the mixture of healthy hairy skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least 5 times as strongly in healthy hairy skin as in hairless skin, or the mixture examined in b) of diseased or hairy skin in disturbed homeostasis arranges if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are expressed at least 5 times as strongly in hair
  • a method for determining the homeostasis of hairy skin is characterized in that in step b) the mixture obtained is examined for the presence and, if appropriate, the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which are shown in Table 3 defined in column 7 by their UniGene accession number, in step c) comparing the test results from b) with the expression quotients given in table 3 in column 3 and column 5 and in step d) the mixture of healthy hairy skin examined in b) assigned if it contains predominantly proteins, mRNA molecules or fragments of proteins or mRNA molecules that are at least 10- in healthy hairy skin are expressed as strongly as in hairless skin, or assigns the mixture examined in b) to diseased or hairy skin in disturbed homeostasis if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are in hairless skin expressed at least 10 times as strongly as in hairy skin.
  • condition of the skin can also be described by quantifying a number of markers (expression products of the genes which are important for hairy skin), which must then be active in a characteristic ratio to one another in order to represent healthy (inostasis) hairy skin, or must be active in a characteristic ratio different from this in order to represent diseased skin (which is in disturbed homeostasis).
  • markers expression products of the genes which are important for hairy skin
  • Another object of the present invention is therefore a method j3) for determining the homeostasis of hairy skin in humans, in particular women, in vitro, which is characterized in that a) a mixture of proteins, mRNA molecules or fragments of proteins or mRNA molecules from hairy human skin, b) quantified in the mixture obtained at least two of the proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are identified by method (1) as being important for hairy skin, c) the Expression ratios of the at least two proteins, mRNA molecules or fragments of proteins or mRNA molecules to one another are determined and the expression quotient is formed, d) comparing the expression ratios from c) with the expression ratios which are typically hairy or in for the molecules quantified in b) hairless skin, especially with the expression ratios, d ie from tables 3 to 12, columns 3 and 5, respectively, and e) assigns the mixture obtained in a) to healthy (in homeostasis) hairy skin if the expression ratios of
  • the mixture is preferably obtained from a skin sample, in particular from a whole skin sample or from an epidermis sample.
  • a skin sample in particular from a whole skin sample or from an epidermis sample.
  • the ⁇ Völlh ütprbbe " Cimfussihdere TM opens up possibilities for comparison ⁇ with the SAGE libraries, which are also obtained from whole skin.
  • the epidermis sample on the other hand, is easier to obtain, for example by applying an adhesive tape to the skin and tearing it off, as described in WO 00/10579 , to which reference is hereby made in full.
  • the mixture is obtained in step a) by means of microdialysis.
  • microdialysis The technique of microdialysis is described, for example, in “Microdialysis: A method for measurement of local tissue metabolism", Nielsen PS, Winge K, Petersen LM; Ugeskr Laeger 1999 Mar 22 161: 12 1735-8; and in “Cutaneous microdialysis for human in vivo dermal absorption studies ", Anderson, C. et al. ; Drugs Pharm. Sci., 1998, 91, 231-244; and also described on the Internet at http://www.microdialysis.se/techniqu.htm, to which reference is hereby made in full.
  • 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 hairy skin is characterized in that in step b) in method (2) the examination for the presence and optionally the amount of at least one of the proteins or protein fragments; or in method (3) the quantification of at least two proteins or protein fragments is carried out by means of a method which is selected from ii. Affinity Chromatography iii. Protein-protein complexation in solution iv. Mass spectrometry, especially matrix assisted laser desorption ionization (MALDI) and especially v. Use of protein chips, or by means of suitable combinations of these methods.
  • MALDI matrix assisted laser desorption ionization
  • 2D gel electrophoresis is described, for example, in L.D. Adams, Two-dimensional Gel Electrophoresis using the Isodalt System or in L.D. Adams & S.R. Gallagher, Two-dimensional Gel Electrophoresis using the O'Farrell System; both in Current Protocols in Molecular Biology (1997, Eds. F.M. Ausubel et al.), Unit 10.3.1 - 10.4.13; or in 2-D electrophoresis manual; T. Berkelman, T. Senstedt; Amersham Pharmacia Biotech, 1998 (order no. 80-6429-60).
  • the mass spectrometric characterization of the proteins or protein fragments is carried out in a manner known to those skilled in the art, for example as described in the following references: Methods in Molecular Biology, 1999; Vol 112; 2-D Proteome Analysis Protocols; Editor: AJ Link; Humana Press; Totowa; New Jersey. In particular: Courchesne, PL and Patterson, SD; Pp. 487-512.
  • Determination of the homeostasis of hairy skin is characterized in that in step b) in method (2) the examination for the presence and, if appropriate, the amount of at least one of the mRNA molecules or mRNA molecule fragments; or in method (3) the quantification of at least two mRNA molecules or mRNA molecule fragments is carried out by means of a method which is selected from Northern blnts.
  • 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
  • step b) the presence and optionally the amount of 1 to about 5000, preferably 1 to about 1000, in particular about 10 to about 500, preferably about 10 to about 250, particularly preferably about 10 to about 100 and very particularly preferably about 10 to about 50 of the proteins, mRNA molecules or fragments of proteins or mRNA molecules investigated by their in Tables 3 to 12 in column 7 UniGene Accession Number can be defined.
  • the present invention further provides a test kit for determining the homeostasis of hairy skin in humans in vitro, comprising means for carrying out the method according to the invention for determining the homeostasis of hairy skin.
  • Another object of the present invention is a biochip for determining the homeostasis of hairy skin in humans in vitro, comprising i. a solid, ie rigid or flexible support and ii. on this immobilized probes which are capable of specific binding to at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which are defined in Tables 3 to 12 in column 7 by their UniGene Accession Number.
  • a biochip which is particularly preferred according to the invention comprises probes which are selected from those which specifically bind to at least one
  • Molecules are capable, which are defined in Tables 3 to 6 in column 1 by the following serial number: 2, 4, 9, 12, 14, 16, 22, 25, 29, 31, 35, 36, 38, 39, 40 , 42, 43, 44, 46, 59, 62, 63, 65, 67, 68, 69, 74.
  • a BioChip is a miniaturized functional element with molecules immobilized on a surface, in particular biomolecules, which can serve as specific interaction partners.
  • 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. Compared to these conventional methods, in which only a few genes are analyzed, DNA chip technology allows a few hundred to several tens of thousands of genes to be examined in parallel.
  • a DNA chip essentially consists of a carrier material (e.g. glass or plastic) on which single-stranded, gene-specific probes are immobilized in a high density at a defined location (spot). The technique of probe application and the chemistry of probe immobilization are considered problematic.
  • 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.
  • DNA molecules already present can also be bound to surfaces of support material.
  • the DNA probes can be applied to a carrier using a so-called “pin spotter”.
  • a pin spotter thin metal needles, for example with a diameter of 250 ⁇ m, are immersed in probe solutions and then transfer the attached sample material with defined volumes to the carrier material of the DNA Crisps.
  • the probe is preferably applied by means of a piezocontrolled nanodispenser, which, like an inkjet printer, applies probe solutions with a volume of 100 picoliters to the surface of the carrier material without contact.
  • the probes are immobilized, for example, as described in EP-A-0 965 647:
  • the generation of DNA probes is carried out by means of PCR using a sequence-specific pair of primers, a primer at the 5 'end being modified and a linker with a free one Amino group carries. This ensures that a defined strand of the PCR products can be bound to a glass surface which has been treated with 3-aminopropyltrimethoxysilane and then with 1,4-phenyldiisothiocyanate.
  • the Gene-specific PCR products should ideally have a defined nucleic acid sequence with a length of 200-400 bp and contain non-redundant sequences.
  • mRNA from two to NeT EiseneTTäeTrZellpopulä ⁇ mRN ⁇ s are converted into cDNA by means of " reverse transcription using, for example, fluorescence-labeled nucleotides.
  • the samples to be compared are labeled with, for example, red or green fluorescent nucleotides.
  • the cDNAs are labeled then hybridized with the gene probes immobilized on the DNA chip and subsequently quantified the bound fluorescence.
  • the analysis chips mentioned in DE-A-100 28 257.1-52 and in DE-A-101 02 063.5-52 are particularly preferred for the production of small (up to about 500 probes) biochips.
  • These analysis chips have an electrically addressable structure which allows the samples to be electrofocused. This advantageously makes it possible to focus and immobilize samples regardless of their viscosity with the aid of electrodes at defined points on a grid of points (arrays). Due to the focusing ability, the local concentration of the samples is increased and thus a higher specificity.
  • 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.
  • biochip according to the invention comprises peptide or protein probes, in particular antibodies.
  • Another object of the present invention is the use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are defined in Tables 3 to 12 in column 7 by their UniGene accession number, as markers of hairy skin in humans ,
  • CDT6 whose role as a hair cycle marker is particularly surprising since it would have been more likely that VEGF or angiopoietin could be used as a marker.
  • the DPPIV family and the DNA helicases are particularly noticeable.
  • a role in the hair cycle was also not foreseeable by the expert.
  • Another object of the present invention is a test method for demonstrating the effectiveness of cosmetic or pharmaceutical active ingredients against diseases or impairments of the homeostasis of hairy skin in vitro, characterized in that a) the skin status of hairy skin by an inventive method for determining the homeostasis of hairy skin, or by means of a test kit according to the invention for determining the homeostasis of hairy skin, or by means of a biochips according to the invention determines "&) a drug counter --i!
  • test method according to the invention can be carried out with whole skin samples, hairy skin equivalents, isolated hair follicles, hair follicle equivalents or cells with hairy skin.
  • Another object of the present invention is a test kit for demonstrating the effectiveness of cosmetic or pharmaceutical active substances against diseases or impairments of the homeostasis of hairy skin, comprising means for carrying out the test method according to the invention.
  • Another object of the present invention is the use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are defined in Tables 3 to 12 in column 7 by their UniGene Accession Number, for the detection of the effectiveness of cosmetic or pharmaceutical active substances against diseases or impairments of the homeostasis of hairy skin.
  • Another object of the present invention is a screening method for the identification of cosmetic or pharmaceutical active substances against diseases or impairments of the homeostasis of hairy skin in vitro, which is characterized in that a) the skin status of hairy skin by an inventive method for determining the homeostasis hairy skin, or by a ⁇ rfindungsdorfen- -estrKits-for-determination _ _ der ⁇ omeostase behaarte ⁇ "skin, or determined by means of a biochip according to the invention, b) a potential drug for diseases or disturbances of homeostasis the scalp once or more is applied to the skin, c) the skin status of hairy skin is determined by a method according to the invention for determining the homeostasis of hairy skin, or by means of a test kit according to the invention for determining the homeostasis of hairy skin, or by means of a biochip according to the invention, and d) effective active ingredient ffe determined by comparing the results from a) and
  • Another object of the present invention is the use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are defined in Tables 3 to 12 in column 7 by their UniGene Accession Number, for the identification of cosmetic or pharmaceutical Active substances against diseases or impairments of the homeostasis of hairy skin.
  • Another object of the present invention is a method for producing a cosmetic or pharmaceutical preparation against diseases or impairments of the homeostasis of hairy skin, characterized in that a) active ingredients using the screening method according to the invention, or the use for the identification of cosmetic or active pharmaceutical ingredients against diseases or
  • Impairments in the homeostasis of hairy skin are determined and active ingredients found to be effective are mixed with cosmetically and pharmacologically suitable and compatible carriers.

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Abstract

La présente invention concerne un procédé de détermination in vitro de l'homéostasie de peau pileuse, des ensembles de test, des puces biologiques destinées à la détermination de marqueurs de peau pileuse, 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 peau pileuse. L'invention concerne également un procédé de test destiné à la détection de l'efficacité d'agents cosmétiques ou pharmaceutiques destinés au traitement de peau pileuse, un procédé de criblage destiné à l'identification d'agents cosmétiques ou pharmaceutiques destinés au traitement de peau pileuse, ainsi qu'un procédé de fabrication d'une composition cosmétique ou pharmaceutique destinée au traitement de peau pileuse.
EP03767789A 2002-12-20 2003-12-11 Procede de determination de l'homeostasie de peau pileuse Withdrawn EP1573053A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10260931 2002-12-20
DE10260931A DE10260931B4 (de) 2002-12-20 2002-12-20 Verfahren zur Bestimmung der Homeostase behaarter Haut
PCT/EP2003/014070 WO2004059002A2 (fr) 2002-12-20 2003-12-11 Procede de determination de l'homeostasie de peau pileuse

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EP1573053A2 true EP1573053A2 (fr) 2005-09-14

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DE10340373A1 (de) * 2003-08-30 2005-03-24 Henkel Kgaa Verfahren zur Bestimmung von Haarzyklus-Marken
DE102005011957A1 (de) * 2005-03-14 2006-12-07 Henkel Kgaa Neues Haarbehandlungsmittel, enthaltend L-Carnitin oder L-Carnitinderivate
FR2948021A1 (fr) * 2009-07-16 2011-01-21 Oreal Utilisation cosmetique de polypeptides de type lacritine
CA2798517C (fr) 2010-05-17 2016-07-05 The Procter & Gamble Company Systemes et procedes de detection et d'identification d'un dommage capillaire par le biais de l'evaluation de fragments proteiques
DE102011079661A1 (de) * 2011-07-22 2013-01-24 Henkel Ag & Co. Kgaa Verfahren zur individualisierten Haarbehandlung
US11365333B2 (en) 2016-05-09 2022-06-21 The Trustees Of The University Of Pennsylvania Omni-transparent and superhydrophobic coatings assembled from chain-like nanoparticles
NO344051B1 (en) * 2017-05-04 2019-08-26 Patogen As Novel virus in Fish and Method for detection

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US6013445A (en) * 1996-06-06 2000-01-11 Lynx Therapeutics, Inc. Massively parallel signature sequencing by ligation of encoded adaptors
US5866330A (en) * 1995-09-12 1999-02-02 The Johns Hopkins University School Of Medicine Method for serial analysis of gene expression
AU4771199A (en) * 1998-06-10 1999-12-30 Memorec Medical Molecular Research Cologne Stoffel Gmbh Support for the parallel identification and establishment of transcription profiles of polynucleic acids
DE10100127A1 (de) * 2001-01-03 2002-10-02 Henkel Kgaa Verfahren zur Bestimmung der Homeostase der Haut
DE10100121A1 (de) * 2001-01-03 2002-08-01 Henkel Kgaa Verfahren zur Bestimmung des Hautstreß oder der Hautalterung in vitro
DE10102063A1 (de) * 2001-01-17 2002-07-25 Alpha Technology Ges Fuer Ange Analysechip mit mehreren funktionalen Ebenen für elektrofokussiertes Spotten

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WO2004059002A2 (fr) 2004-07-15
US20060088852A1 (en) 2006-04-27
DE10260931A1 (de) 2004-07-08
WO2004059002A3 (fr) 2005-06-30
AU2003292230A1 (en) 2004-07-22

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