EP1658380A2 - Procede pour determiner des marqueurs de cycle capillaire - Google Patents

Procede pour determiner des marqueurs de cycle capillaire

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Publication number
EP1658380A2
EP1658380A2 EP04764414A EP04764414A EP1658380A2 EP 1658380 A2 EP1658380 A2 EP 1658380A2 EP 04764414 A EP04764414 A EP 04764414A EP 04764414 A EP04764414 A EP 04764414A EP 1658380 A2 EP1658380 A2 EP 1658380A2
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EP
European Patent Office
Prior art keywords
proteins
hair
mrna molecules
column
fragments
Prior art date
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EP04764414A
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German (de)
English (en)
Inventor
Olaf HOLTKÖTTER
Dirk Petersohn
Kordula Schlotmann
Melanie Giesen
Daniela Kessler-Becker
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Henkel AG and Co KGaA
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Henkel AG and Co KGaA
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Application filed by Henkel AG and Co KGaA filed Critical Henkel AG and Co KGaA
Publication of EP1658380A2 publication Critical patent/EP1658380A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a method for determining hair cycle markers in vitro, test kits and biochips for determining hair cycle markers and the use of proteins, mRNA molecules or fragments of proteins or mRNA molecules as hair cycle markers; furthermore a test method for the detection of the effectiveness of cosmetic or pharmaceutical active ingredients for influencing the hair cycle as well as a screening method for the identification of cosmetic or pharmaceutical active ingredients for influencing the hair cycle and a method for producing a cosmetic or pharmaceutical preparation for influencing the hair cycle.
  • Hair follicles go through a cycle of three stages: anagen (growth phase), catagen (regression phase) and telogen (resting phase).
  • growth phase growth phase
  • catagen progression phase
  • telogen resting phase
  • androgenic alopecia is characterized by a shorter anagen phase combined with a reduction in the size of the hair follicle (e.g. Paus and Cotsarelis (1999), New Eng. J. Med., 341: 491-497).
  • the hair follicle is assigned to a stage of the hair cycle essentially based on the microscopic-morphological analysis of the hair.
  • the molecular mechanisms that play a role in the progression through the hair cycle are only poorly understood. Consequently, there are no molecular markers that are characteristic of a particular stage of the hair follicle, just as there are no sufficiently large number of molecular targets that influence the condition of the hair follicle can.
  • a number of different markers of human hairy skin could be identified in the applicant's DE 102 60931.4, these are primarily characteristic of the anagen hair follicles, which make up the largest proportion of hairy skin.
  • transcriptome i.e. the entirety of all transcribed genes, of the hair follicle has not been described in various stages of the cell cycle.
  • 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 the hair cycle 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 to identify as large a part of the genes as are important for the hair cycle.
  • the hair cycle is to be determined by means of the identified genes and methods for finding active substances for influencing the hair cycle are to be provided.
  • This first object is achieved according to the invention by a method (1) for identifying the genes which are important for the hair cycle (hair cycle markers) in humans, characterized in that a) a first mixture of expressed, ie transcribed and in anagenous human hair follicles optionally also translated genetically coded factors, i.e. proteins, mRNA molecules or fragments of proteins or mRNA molecules from hairy human skin, preferably from hairy scalp, b) a second mixture of expressed, ie transcribed and optionally in human hair follicles also translates genetically coded factors, i.e.
  • a first mixture of expressed, ie transcribed and in anagenous human hair follicles optionally also translated genetically coded factors, i.e. proteins, mRNA molecules or fragments of proteins or mRNA molecules from hairy human skin, preferably from hairy scalp
  • a second mixture of expressed, ie transcribed and optionally in human hair follicles also translates genetically coded
  • the method according to the invention advantageously makes it possible to understand the complex process of the hair cycle and the causal relationships of the changes in the hair follicle. 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.
  • SAGE TM analysis carried out as part of the method according to the invention shows for the first time in a very comprehensive manner which genes are expressed differently in anagen and catagen hair follicles.
  • transcriptome The entirety of all mRNA molecules that are synthesized by a cell or a tissue at a specific point in time is referred to as a "transcriptome”.
  • SAGE TM serial analysis of gene expression
  • This technique simultaneously allows the identification and quantification of those expressed in hair follicles
  • the comparison of the transcriptome of anagen hair follicles with the transcriptome of catagen hair follicles allows the identification of relevant genes of the hair cycle, which can be genes that are particularly strongly expressed in anagen hair follicles or genes that are characterized in that they are compared to catagen hair follicles are only slightly expressed.
  • the SAGE method is based on two principles: First, only a short nucleotide sequence from the 3 ' region of the mRNA is required to identify the gene. A sequence of nine base pairs enables differentiation of 262,144 (4 9 ) transcripts. That is more than the number of all genes present in the genome. Secondly, the concatenation of the short sequences enables efficient automated analysis using sequencing. One advantage of this technique that should not be underestimated is the determination of the reading direction of the genes. If two opposite transcripts of a gene are started in the reading direction, this can only be determined with the SAGE technique. Typically, double-stranded cDNA is synthesized with biotinylated primers from polyA-RNA.
  • the cDNA is digested with a 4bp recognizing restriction enzyme (anchoring enzyme), which statistically cut every 256bp.
  • the 3 'end of the cDNA is isolated by binding to streptavidin beads.
  • the sample is divided into two halves and the cDNA end is ligated with a linker (1 or 2), which has a recognition site for a type III restriction enzyme (tagging enzyme). This cuts up to 20bp offset from the asymmetrical detection point. This creates a short sequence (tag) bound to the linker, which is unique for each gene.
  • the linkers! -Tags after filling the protruding ends with the Linker2-7ags ligated (Linkerditag).
  • the ligation products are amplified with linker-specific primers (1 or 2). Then the linker that is no longer used is released by a further enzymatic digestion with the anchoring enzyme.
  • the isolated ditags are lined up (concatemere), cloned into a vector and transfected into cells. The concatemers are amplified from the cells via PCR and finally sequenced.
  • Another promising method is micro-array or chip technology.
  • entire gene libraries are brought together on one chip.
  • the genes on the chip are hybridized with fluorescence-labeled cDNA, which was generated from the mRNA of the tissue sample to be examined.
  • fluorescence-labeled cDNA was generated from the mRNA of the tissue sample to be examined.
  • a very advantageous analysis method is the combination of the SAGE and the M / ⁇ O / 4rray technique.
  • the SAGE method provides new or known genes for the hair cycle can be significant. These are projected onto a chip that can now be used to measure samples from individual candidates.
  • Human hair follicles from healthy female donors were used for SAGE TM analysis.
  • the follicles were isolated from pieces of tissue taken from the donor's ear and sorted according to their morphology according to catagen and anagen hair follicles.
  • the catagen and anagen hair follicles of a total of five donors were combined. The same number of catagen and anagen follicles from a donor was used and the total number of follicles from the individual donors was adjusted to each other.
  • SAGE TM analysis was carried out as in Veiculescu, V.E. et al., 1995 Science 270, 484-487.
  • One SAGE TM bank was analyzed for catagen and one for anagen hair follicles.
  • both SAGE TM banks were normalized to the average number of days. The two banks were compared to identify genes with hair cycle specific regulation.
  • Table 1 lists markers for which differential expression depending on the stage of the hair cycle has already been described. They serve as positive controls for the experiment. Specified are: • the relative expression frequency in anagen hair follicles in column 1, • the relative expression frequency in catagen hair follicles in column 2, • the quotient of the determined relative expression frequency in anagen hair follicles and the determined relative expression frequency in catagen hair follicles in column 3, • the significance of the values mentioned in column 3 in column 4, • the UniGene Accession Number in column 5, • the Swissprot Accession number in column 6 and • the name of the gene from which the corresponding tag originates in column 7
  • 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 anagen hair follicles than in catagen hair follicles, or vice versa.
  • mice whose vitamin D receptor has been inactivated are characterized by hair loss. It could be shown that after stimulation of the anagen stage by shaving, mice with an inactive vitamin D receptor cannot initiate the hair cycle (Kong et al. (2002), J Invest Dermatol, 118: 631-
  • Thrombospondin-1 has been shown to play a role in inducing hair follicle involution and in vascular breakdown during the catagen phase (Yano et al. (2003), J Invest Dermatol, 120: 14-9). While no expression of thrombospondin can be detected in the early to middle anagen phase, a strong expression during the catagen phase is shown, in accordance with the expression data found here.
  • the role of neurotrophin-5 for human hair follicles has not been described so far, but there are studies on the family member neurotrophin-3 in murine hair follicles. Maximum expression of the neurotrophin was observed in the catagenic stage (Botchkarev et al. (1998), Am J Pathol, 153: 785-99). A corresponding expression pattern was found here for neurotrophin-5.
  • Tables 2 to 6 contain a detailed list of the genes determined with the aid of the method according to the invention and differentially expressed in anagen hair follicles and in catagen hair follicles, stating • the current sequence ID (cf. sequence listing) in column 1, • the tag sequence used in column 2, • the relative expression frequency in anagen hair follicles in column 3, • the relative expression frequency in catagen hair follicles in column 4, • the quotient of the determined relative expression frequency in anagen hair follicles and the determined relative expression frequency in catagen hair follicles in column 5, • the Significance of the values in column 5 in column 6, • the UniGene Accession Number in column 7, • the Swissprot Accession Number in column 8 and • a brief description of the gene or gene product in column 9
  • the quotient in column 5 indicates the strength of the differential expression, ie by which factor the respective gene is more strongly expressed in anagen hair follicles than in catagen hair follicles, or vice versa
  • Table 2 lists all genes that are expressed at least 5-fold differentially in anagen hair follicles compared to catagen hair follicles with a p-value of p ⁇ 0.01 (significance> 2.0).
  • Table 3 lists all genes that are at least 2-fold differentially expressed in anagen hair follicles compared to catagen hair follicles with a p-value of p ⁇ 0.01 (significance> 2.0).
  • Table 4 lists all genes that are expressed at least 1.3 times differentially in anagen hair follicles compared to catagen hair follicles with a p-value of p ⁇ 0.01 (significance> 2.0).
  • Table 5 lists all genes which are expressed at least 5-fold differently in anagen hair follicles compared to catagen hair follicles with a p-value of p ⁇ 0.05 (significance> 1, 3).
  • Table 6 lists all genes that are at least 2-fold differentially expressed in anagen hair follicles compared to catagen hair follicles with a p-value of p ⁇ 0.05 (significance> 1, 3).
  • the clear difference in expression of the ribosomal RNAs is particularly striking here. Small differences in expression in ribosomal RNAs have so far been described as typical artifacts of SAGE TM. In the present
  • Attractin is a protein from the agouti / melanocortin
  • the gene product plays a role in determining the
  • mice Hair color of mice (Gunn et al. (1999), Nature, 398: 152-6; Barsh et al. (2002),
  • Adenosyltransferase an enzyme of vitamin B12 metabolism.
  • a vitamin B12 Adenosyltransferase, an enzyme of vitamin B12 metabolism.
  • Dopachrome tautomerase an enzyme in melanin biosynthesis, is also induced in catagenic hair follicles. All of the above genes are relevant for hair follicle biology, especially for pigmentation, but so far not in
  • angiopoietin-like protein CDT6 can be found in the catagenic hair follicles, a protein for which a regulatory
  • Hair follicle is coupled to the hair cycle (see above Thrombospondin-1).
  • the 14-3-3 family of proteins regulate a variety of enzymes, including those of primary metabolism and of the cell cycle. Furthermore, they have a chaperone function, they can activate the transcription of inducible genes and regulate signal transduction and apoptosis processes.
  • a role in the differentiation of keratinocytes has been described in particular for the protein 14-3-3 sigma (stratifin) (Dellambra et al. (1995), J Cell Sei 108: 3569-79). A specific regulation of the members of this protein family in the different hair follicle stages is therefore extremely likely.
  • the keratin 6A and the aeidie hair keratin are also found in catagen hair follicles.
  • Table 7 contains a detailed list of the genes determined with the aid of the method according to the invention and differentially expressed in anagen hair follicles and in catagen hair follicles, stating • the current sequence ID (see sequence listing) in column 1, • the tag sequence used in column 2 , • the relative expression frequency in anagen hair follicles in column 3, • the relative expression frequency in catagen hair follicles in column 4, • the ratio of the determined relative expression frequency in anagen hair follicles and the determined relative expression frequency in catagen hair follicles to each other in column 5, • the significance of the values mentioned in column 5 in column 6, • the GO number in column 7, • a brief description of the gene or gene product in column 8 and • the Swissprot Accession Number in column 9
  • 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 anagen hair follicles than in catagen hair follicles, or vice versa.
  • the dipeptidyl peptidases of the DPP-IV family are proline-specific proteases, the main function of which appears to be the regulation of various pathological and physiological processes (Aleski and Malik
  • Type IV collagen is a typical part of the follicle matrix, and one Increased expression of this protein is expected in the growth phase of the follicle.
  • the "synaptosome” cluster is also induced in the anagen hair follicles.
  • This cluster contains the SNARE proteins VAMP-2 [5: 0] and VAMP-3 [4: 0], which have a general role in secretion.
  • VAMP-2 [5: 0] and VAMP-3 [4: 0] have a general role in secretion.
  • This observation is supported by the general induction of genes that play a role in exocytosis. This induction of exocytosis genes is probably related to the process of pigmentation of the hair. Pigmentation requires the transfer of melanin-synthesizing organelles, so-called melanosomes, from melanocytes to keratinocytes in the hair follicle.
  • Poly-N-acetyllactosamine structures are found in both N- and O-linked glycans of mammalian glycoproteins. These glycans presumably interact with selectins and other glycan-binding proteins (Zhou (2003), Curr Protein Pept Sei, 4: 1-9).
  • clustering is made possible by comparing the SAGE data with the data from available domain and motif databases, for example PROSITE and Pfam [http://www.sanger.ac.uk/Software/Pfam/index.shtml; http://www.expasy.ch/prosite/].
  • Table 8 contains a detailed list of the genes determined with the aid of the method according to the invention and differentially expressed in anagen hair follicles and in catagen hair follicles, stating • the current sequence ID (cf. sequence listing) in column 1, • the tag sequence used in column 2, • the relative expression frequency in anagen hair follicles in column 3, • the relative expression frequency in catagen hair follicles in column 4, • the ratio of the determined relative expression frequency in anagen hair follicles and the determined relative expression frequency in catagen hair follicles to each other in column 5, • the significance of the values mentioned in column 5 in column 6, • a brief description of the motif or gene or gene product in column 7 and • the Swissprot Accession Number in column 8
  • the quotient in column 5 indicates the strength of the differential expression, i. i.e. by which factor the respective gene is expressed more strongly in anagen hair follicles than in catagen hair follicles, or vice versa.
  • the significance of the differential expression of different genes can be increased by lexical analysis.
  • matching keywords in the description texts become the different Genes such as those found in the database annotations were searched for.
  • Table 9 contains a detailed list of the genes determined with the aid of the method according to the invention and differentially expressed in anagen hair follicles and in catagen hair follicles, stating • the current sequence ID (see sequence listing) in column 1, • the tag sequence used in column 2 , • the relative expression frequency in anagen hair follicles in column 3, • the relative expression frequency in catagen hair follicles in column 4, • the ratio of the determined relative expression frequency in anagen hair follicles and the determined relative expression frequency in catagen hair follicles to each other in column 5, • the significance of the values in column 5 in column 6, • the search word in column 7, • a short description of the gene or gene product in column 8 and • the Swissprot Accession Number in column 9
  • 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 anagen hair follicles than in catagen hair follicles, or vice versa.
  • Autophagy is a process in which cells envelop macroscopic cell components, such as organelles, in autophagosomes and then digest them in the lysosome. Autophagy mainly occurs when there is a lack of cells; Excessive autophagy is considered a mechanism of non-apoptotic programmed cell death. Furthermore, clusters are repressed in catagen hair follicles, which are formed on the basis of the keywords “dsc2” and “desmocollin”.
  • the second object underlying the present invention is achieved according to the invention by a method (2) for determining the hair cycle 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 or from human hair follicles, b) the mixture obtained for the presence and possibly the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules examined by means of serial analysis gene expression (SAGE) are identified as being differentially expressed in anagen and catagenic human hair follicles, c) comparing the test results from b) with the expression patterns identified by means of serial analysis of gene expression (SAGE) and d) the growing or healthy mixture examined in b) Allocates hair when it's predominant Contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are more strongly expressed in anagen hair follicles than in catagen hair follicles, or which the mixture examined in
  • the mixture in step a) of the method according to the invention for determining the hair cycle 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 hair cycle 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 gene expression (SAGE) identified as differentially expressed in anagen and catagen hair follicles if these are expressed exclusively in anagen or only in catagen hair follicles.
  • SAGE serial analysis of gene expression
  • step d) of the method for determining the hair cycle the mixture examined in b) is assigned to growing or healthy hair if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are more strongly expressed in anagen hair follicles than in catagens, that is, the mixture either contains more different compounds typically expressed in anagen hair follicles than those typically expressed in catagen hair follicles (qualitative differentiation), or contains more copies of compounds typically expressed in anagen hair follicles than typically in catagenes Hair follicles are present (quantitative differentiation). For the assignment to hair in regression or sick hair is used in a complementary manner.
  • a preferred embodiment of the method according to the invention for determining the hair cycle 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 Table 9 in column 9 by their Swissprot Accession Number and in step d) assigns the mixture examined in b) to growing or healthy hair if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed more strongly in anagen hair follicles than in catagen, or which assigns the mixture examined in b) to regression or diseased hair if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are present in catagen hair follicles are expressed more than in anagen.
  • a further preferred embodiment of the method according to the invention for determining the hair cycle 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 Table 8 in column 8 by their Swissprot Accession Number and in step d) assigns the mixture examined in b) to growing or healthy hair if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules contains which are expressed more strongly in anagen hair follicles than in catagen, or which assigns the mixture examined in b) to regression or diseased hair if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are in catagen Hair follicles are expressed more than in anagen.
  • 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 Table 7 in column 9 by their Swissprot Accession Number and in step d) assigns the mixture examined in b) to growing or healthy hair if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are more strongly expressed in anagen hair follicles than in catagen, or that in b ) Examines the mixture under regression or diseased hair if it contains ' predominantly proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed more strongly in catagenic hair follicles than in anagenic ones.
  • a further preferred embodiment of the method according to the invention for determining the hair cycle 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 Table 6 in column 7 by their UniGene Accession Number, in column 8 by their Swissprot Accession Number, or in column 9 by their short description of the gene or gene product and in step d) that examined in b) Mixes growing or healthy hair if it contains predominantly proteins, mRNA molecules or fragments of proteins or mRNA molecules that are expressed in anagen hair follicles at least twice as strongly as in catagen, or the mixture examined in b) is in regression or sick hair if it predominantly contains proteins, mRNA molecules or fragments of proteins contains n or mRNA molecules that are expressed in catagenic hair follicles at least twice as strongly as in anagenic ones.
  • a further preferred embodiment of the method according to the invention for determining the hair cycle is characterized in that in step b) the mixture obtained is checked for the presence and optionally the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which are defined in Table 5 in column 7 by their UniGene accession number, in column 8 by their Swissprot accession number, or in column 9 by their short description of the gene or gene product and in Step d) assigns the mixture examined in b) to growing or healthy hair if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least 5 times as strongly in anagen hair follicles as in catagen, or assigns the mixture examined in b) to regression or diseased hair if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are expressed at least 5 times as strongly in catagen hair follicles as in anagen.
  • 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 Table 4 in column 7 by their UniGene Accession Number, in column 8 by their Swissprot Accession Number, or in column 9 by their short description of the gene or gene product and in step d) that in b) Allocated mixture assigned to growing or healthy hair if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are expressed at least 1.3 times as strongly in anagen hair follicles as in catagen, or that in b) investigated mixture assigned to regression or diseased hair if it predominantly contains proteins, mRNA molecules or fragments contains proteins or mRNA molecules that are expressed in catagen hair follicles at least 1.3 times as strongly as in anagen.
  • step b) the mixture obtained is examined for the presence and, if appropriate, the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are listed in table 3 in column 7 by their UniGene Accession Number, in column 8 are defined by their Swissprot Accession Number, or in column 9 by their short description of the gene or gene product and in step d) assigns the mixture examined in b) to growing or healthy hair if it predominantly contains proteins, mRNA molecules or includes fragments of proteins or mRNA molecules which are in anagen 'hair follicles at least 2 times as strongly expressed, as in catagen, or examined in b) mixture assigns in regression befindlichem or diseased hair when it predominantly proteins, mRNA molecules or contains fragments of proteins or mRNA molecules that are expressed at least twice as strongly in catagenic hair follicles as in anagenic ones.
  • 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 7 by their UniGene Accession Number, in column 8 by their Swissprot Accession Number, or in column 9 by their short description of the gene or gene product and in step d) that in b ) investigates the mixture of growing or healthy hair if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are expressed at least 5 times as strongly in anagen hair follicles as in catagen, or that was examined in b) Mixture in regression or diseased hair if it predominantly contains proteins, mRNA molecules or fragments contains proteins or mRNA molecules which are expressed at least 5 times as strongly in catagen hair follicles as in anagen.
  • the hair cycle can also be described in that several markers (expression products of the genes which are important for anagen or catagen hair follicles) are quantified, which must then be active in a characteristic ratio to one another in order to represent growing or healthy hair, or in one of them different characteristic ratios must be active in order to represent regression or diseased hair.
  • the present invention therefore furthermore relates to a method (3) for determining the hair cycle 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 or from human hair follicles, 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 important for the hair cycle, 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) the expression ratios from c) are compared with the expression ratios which are typically found in anagen for the molecules quantified in b) or present in catagen hair follicles, especially with the Expression ratios that result from Tables 2 to 6, column 5, and e) assign the mixture obtained in a) to growing or healthy hair, if the expression ratio
  • 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 is characterized in that in step b) in method (2) the examination for the presence and optionally the amount of at least one of the proteins or protein fragments; or in method (3) the quantification of at least two proteins or protein fragments is carried out by means of a method which is selected from one- or two-dimensional gel electrophoresis affinity chromatography protein-protein complexation in solution iv. Mass spectrometry, especially matrix assisted laser desorption ionization (MALDI) and especially v. Use of protein chips, or by means of suitable combinations of these methods.
  • 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).
  • a further preferred embodiment of the method according to the invention 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 i. Northern Blots, ii. Reverse transcriptase polymerase chain reaction (RT-PCR), iii. RNase protection experiments, iv. Dot blots, v. cDNA sequencing, vi. Clone hybridization, vii. Differential display, viii. Subtractive hybridization, ix. cDNA fragment fingerprinting, x.
  • RT-PCR Reverse transcriptase polymerase chain reaction
  • iii. RNase protection experiments iv. Dot blots, v. cDNA sequencing, vi. Clone hybridization, vii. Differential display, viii. Sub
  • TOGA Total Gene Expression Analysis
  • xi Serial analysis of gene expression
  • xii Massively Parallel Signature Sequencing
  • MPSS ® Massively Parallel Signature Sequencing
  • the MPSS ® process is US Patent No. 6,013,445 described in, which are hereby fully comprehensive cover.
  • a further preferred embodiment of the processes according to the invention is characterized in that in step b) the presence and optionally the amount of from 1 to about 5000, preferably from 1 to about 1000, in particular from about 10 to about 500, preferably from 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 listed in Table 8 in column 8 by their Swissprot Accession Number, in the tables 7 and 9 in column 9 by their Swissprot Accession Number and in Tables 2 to 6 in column 7 by their UniGene Accession Number, in column 8 by their Swissprot Accession Number, or in column 9 by their short description of the Gene or gene product can be defined.
  • the present invention further provides a test kit for determining the hair cycle in humans in vitro, comprising means for carrying out the method according to the invention for determining the hair cycle in humans.
  • Another object of the present invention is a biochip for determining the hair cycle in humans in vitro, comprising i. a firm, d. H. rigid or flexible beams and ii. on this immobilized probes, which are capable of specific binding to at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are listed in Table 8 in column 8 by their Swissprot Accession Number, in Tables 7 and 9 in column 9 by their Swissprot accession number and in Tables 2 to 6 in column 7 by their UniGene accession number, in column 8 by their Swissprot accession number, or in column 9 by their short description of the gene or Gene product to be defined.
  • 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.
  • DNA molecules already present 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 contact 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 converted into cDNA by means of reverse transcription using, for example, fluorescence-labeled nucleotides.
  • the samples to be compared are marked with, for example, red or green fluorescent nucleotides.
  • the cDNAs are then hybridized with the gene probes immobilized on the DNA chip and the bound fluorescence is then quantified.
  • a key success factor for using DNA chip technology to analyze gene expression in hair follicles is the composition of the gene-specific probes on the DNA chip.
  • the relevant genes of the hair cycle identified in the SAGE TM analysis are used.
  • RNA chip Since the use of a DNA chip to analyze the relevant genes of the hair cycle sometimes requires extremely small amounts of mRNA to be examined, it may become necessary to analyze the mRNA prior to analysis using so-called linear amplification (Zhao et al. (2002) , BMC Genomics, 3:31).
  • the mRNA of a sample is first rewritten into cDNA.
  • the amplified RNA is obtained from this double-stranded cDNA by in vitro transcription.
  • 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.
  • a DNA chip is particularly preferred which has probes selected from the probes listed in Tables 2 and 5, the probes listed in Table 3 (without mitochondrial and ribsomal tags and the overrepresented groups "DNA helicase activity”, “DPPIV activity” and “Melanin biosynthesis from tyrosine” from Table F.
  • the biochip according to the invention comprises peptide or protein probes, in particular antibodies.
  • Another object of the present invention is the use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are listed in Table 8 in column 8 by their Swissprot Accession Number, in Tables 7 and 9 in column 9 by their Swissprot-Accession-Number and in Tables 2 to 6 in column 7 by their UniGene-Accession-Number, in column 8 by their Swissprot-Accession-Number, or in column 9 by their short description of the gene or gene product Hair cycle markers in humans.
  • Another object of the present invention is a test method for detecting the effectiveness of cosmetic or pharmaceutical active ingredients for influencing the hair cycle, in particular against diseases or impairments of the hair and their growth, in vitro, characterized in that a) the hair status by a method according to the invention for Determination of the hair cycle, or by means of a test kit according to the invention for determining the hair cycle, or by means of a biochip according to the invention, b) applying an active ingredient against diseases or impairments of the hair and its growth to the hairy skin one or more times, c) again the hair status by a method according to the invention for determining the hair cycle, or by means of a test kit according to the invention for determining the hair cycle, or by means of of a biochip according to the invention is determined, and d) the effectiveness of the active ingredient is determined by comparing the results from a) and c).
  • 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 hair and their growth, 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 listed in Table 8 in column 8 by their Swissprot Accession Number, in Tables 7 and 9 in column 9 by their Swissprot-Accession-Number and in Tables 2 to 6 in column 7 by their UniGene-Accession-Number, in column 8 by their Swissprot-Accession-Number, or in column 9 by their short description of the gene or gene product Proof of the effectiveness of cosmetic or pharmaceutical active substances against diseases or impairments of the hair and their growth.
  • 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 hair and their growth in vitro, which is characterized in that a. determines the hair status by a method according to the invention for determining the hair cycle, or by means of a test kit according to the invention for determining the hair cycle, or by means of a biochip according to the invention, b) applying a potential active ingredient against diseases or impairments of the hair and its growth to the hairy one or more times Applies skin, c) determines the hair status by a method according to the invention for determining the hair cycle, or by means of a test kit according to the invention for determining the hair cycle, or by means of a biochip according to the invention, and d) active ingredients by comparing the results from a) and c ) certainly.
  • Another object of the present invention is the use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are listed in Table 8 in column 8 by their Swissprot Accession Number, in Tables 7 and 9 in column 9 by their Swissprot-Accession-Number and in Tables 2 to 6 in column 7 by their UniGene-Accession-Number, in column 8 by their Swissprot-Accession-Number, or in column 9 by their short description of the gene or gene product Identification of cosmetic or pharmaceutical active ingredients against diseases or impairments of the hair and its growth.
  • Another object of the present invention is a method for producing a cosmetic or pharmaceutical preparation against diseases or impairments of the hair and its growth, characterized in that a) active ingredients with the help 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 hair and their growth and b) active ingredients found to be effective are mixed with cosmetically and pharmacologically suitable and compatible carriers.
  • neurotrophin 4 AnagekatageriQuotienfeignifikan UniGene Swissproi Tag ID 7.98 5.01 1.59 0.37 Hs. 2062 P11473 vitamin D receptor 1.00 2.00 -2.00 0.60 Hs. 87409 P07996 thrombospondin 1 1.00 3.01 - 3.01 0.43 Hs. 26690 P34130 neurotrophin 5 (neurotrophin 4

Abstract

La présente invention concerne un procédé pour déterminer des marqueurs de cycle capillaire in vitro, des kits d'essai et des biopuces pour déterminer des marqueurs de cycle capillaire, ainsi que l'utilisation de protéines, de molécules d'ARNm, de fragments de protéines ou de fragments de molécules d'ARNm en tant que marqueurs de cycle capillaire. L'invention a également pour objet un procédé d'essai pour mettre en évidence l'efficacité de principes actifs cosmétiques ou pharmaceutiques, à agir sur le cycle capillaire, ainsi qu'un procédé de criblage pour identifier des principes actifs cosmétiques ou pharmaceutiques qui agissent sur le cycle capillaire, et un procédé pour préparer une préparation cosmétique ou pharmaceutique qui agit sur le cycle capillaire.
EP04764414A 2003-08-30 2004-08-24 Procede pour determiner des marqueurs de cycle capillaire Withdrawn EP1658380A2 (fr)

Applications Claiming Priority (2)

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DE10340373A DE10340373A1 (de) 2003-08-30 2003-08-30 Verfahren zur Bestimmung von Haarzyklus-Marken
PCT/EP2004/009435 WO2005028671A2 (fr) 2003-08-30 2004-08-24 Procede pour determiner des marqueurs de cycle capillaire

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DE102006042244A1 (de) * 2006-09-06 2008-03-27 Henkel Kgaa Verfahren zur molekularen Charakterisierung von Altershaar
WO2009086000A2 (fr) * 2007-12-19 2009-07-09 Aderans Research Institute, Inc. Biomarqueurs pour trichogénicité
KR101349739B1 (ko) 2012-04-18 2014-01-16 경북대학교 산학협력단 남성형 탈모 진단용 바이오마커 조성물 및 이를 이용한 진단 방법

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US6013445A (en) * 1996-06-06 2000-01-11 Lynx Therapeutics, Inc. Massively parallel signature sequencing by ligation of encoded adaptors
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