WO2002054078A2 - Procede de determination in vitro du vieillissement de la peau - Google Patents

Procede de determination in vitro du vieillissement de la peau Download PDF

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Publication number
WO2002054078A2
WO2002054078A2 PCT/EP2001/014812 EP0114812W WO02054078A2 WO 2002054078 A2 WO2002054078 A2 WO 2002054078A2 EP 0114812 W EP0114812 W EP 0114812W WO 02054078 A2 WO02054078 A2 WO 02054078A2
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Prior art keywords
skin
proteins
stress
fragments
hsp27
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PCT/EP2001/014812
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German (de)
English (en)
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WO2002054078A3 (fr
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Dirk Petersohn
Günter Schmitt
Thomas Förster
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Henkel Kommandigesellschaft Auf Aktien
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Priority to AU2002219190A priority Critical patent/AU2002219190A1/en
Publication of WO2002054078A2 publication Critical patent/WO2002054078A2/fr
Publication of WO2002054078A3 publication Critical patent/WO2002054078A3/fr

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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
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • 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 skin stress and / or skin aging in humans in vitro, test kits and biochips for determining skin stress and / or skin aging and the use of proteins, mRNA molecules or fragments of proteins or mRNA Molecules as skin stress and / or aging markers; furthermore a test method for proving the effectiveness of cosmetic or pharmaceutical active substances against skin stress and / or skin aging as well as a screening method for the identification of cosmetic or pharmaceutical active substances against skin stress and / or skin aging and a method for the production of a cosmetic or pharmaceutical preparation against skin stress and / or or skin aging.
  • the human skin is a very complex organ, which consists of a large number of different cell types.
  • the metabolism of living cells is not static but very dynamic.
  • cells notice changes in their environment (e.g. sunlight) and react to this by changing their RNA and / or protein synthesis performance.
  • Some molecules are increasingly synthesized after a stress stimulus (e.g. sunlight) (e.g. MMP-1), others are produced to a lesser extent (e.g. collagen cti (I)). Furthermore, there will be no significant change in a large number of the synthesis processes (e.g. TIMP-1).
  • a stress stimulus e.g. sunlight
  • MMP-1 e.g. MMP-1
  • others are produced to a lesser extent (e.g. collagen cti (I)).
  • collagen cti (I) e.g. collagen cti (I)
  • the reactions of the skin to stress should not be viewed as reactions of isolated, isolated skin cells. Rather, every cell is integrated into a complex communication network.
  • This network includes, for example, the communication between cells of the epidermis and cells of the dermis.
  • Signal molecules such as interleukins, growth factors (e.g. KGF, EGF or FGF) etc. are involved in the communication between the cells of the skin.
  • the macroscopic phenomena of aging skin are based on the one hand on intrinsic or chronological aging (skin aging), and on the other hand on extrinsic aging due to environmental stress (skin stress).
  • the ability of living skin cells to react to your environment changes over time - there are aging processes that lead to senescence and ultimately cell death.
  • the visible signs of aged skin are to be understood as an integral of intrinsic and extrinsic aging (eg due to sunlight), the events of extrinsic aging accumulating in the skin over a longer period of time.
  • These individual events can be direct reactions of skin cells to the stress stimulus or indirect reactions due to a signal (e.g. interleukins or growth factors) from neighboring, stressed cells.
  • a signal e.g. interleukins or growth factors
  • each cell type of the skin synthesizes approximately 15,000 different proteins. So far it is largely unclear which proteins play a role in intrinsic and / or extrinsic skin aging. To make matters worse, the skin consists of several different cell types (fibroblasts, keratinocytes in different differentiation states, melanocytes, Langerhans cells, hair follicle cells, sweat gland cells, etc.), and the complexity of the protein mixtures produced in the skin is therefore very great.
  • a major problem in the identification of markers of skin aging is the complex composition of the skin from different cell types. This problem can be minimized by restricting yourself to the identification of skin-relevant age markers to individual cell types of the skin. However, it is ignored that cells that were not directly exposed to a stress stimulus age extrinsically due to the communication with neighboring, stressed cells.
  • methods for determining skin stress and / or skin aging are to be provided by means of the identified markers or genes.
  • This first object is achieved according to the invention by a method for identifying markers that are important for skin aging and / or skin stress in humans, characterized in that:
  • a) exposes human skin, a whole skin model or genetically humanized skin of an animal to a stress stimulus, b) a first mixture of translated genetically coded factors, i.e. proteins or fragments of proteins from the skin stressed in a), c) a second mixture of translates genetically encoded factors, i.e. proteins or fragments of proteins from unstressed human skin, an unstressed full skin model or unstressed genetically humanized skin of an animal, d) the mixtures obtained in b) and c) of an analysis of the translated genetically encoded factors subjects, and by comparing the results of this analysis identifies the factors that are produced to different degrees in stressed or old and undressed or young skin.
  • step a) full-thickness skin model is available, for example from the company Cell Systems ® Biotechnologymaschine GmbH of D-53562 St. Catherine under the name Skin Test System AST-2000.
  • a suitable full skin model consists of dermal and epidermal components and is therefore physiologically comparable to natural skin.
  • the skin equivalent consists of dermal fibroblasts (subcutaneous tissue) and a multilayer epidermis with a callus layer (stratum corneum). This skin model comes very close in structure and function to the living, natural skin.
  • Suitable stress stimuli can be, for example: UV light, in particular UV-A (320-400 nm), for example in a dose of approximately 10 J / cm 2 or UV-B (280-320 nm), for example in a dose of approximately 360 mJ / cm 2
  • Sunlight basically the entire solar radiation spectrum.
  • a sub-spectrum can also be used for quantification, for example by o irradiation with sunlight corresponding to a dose of UV-A of approximately 10 J / cm 2 or o irradiation with approximately A to ⁇ 1.0 MED (minimum erythemal dose)
  • the analysis in d) is preferably carried out by means of one- or two-dimensional gel electrophoresis and subsequent mass spectrometry, in particular matrix-assisted laser desorption ionization (MALDI).
  • MALDI matrix-assisted laser desorption ionization
  • the proteins stratifin (14-3-3, ⁇ -isoform), HSP27 (heat shock protein 27 kDa) and GRP78 / BiP are produced by human skin in a stress-inducible manner, ie. H. Stressed skin produces these proteins in a significantly larger amount than non-stressed skin.
  • stressed skin produces phosphorylated, in particular hyperphosphorylated, isoforms of HSP27 (Heat shock protein 27 kDa) in a considerably larger amount than non-stressed skin.
  • the second object on which the present invention is based is therefore achieved according to the invention by a method for determining skin stress and / or skin aging in humans in vitro, characterized in that a) a mixture of proteins or fragments of proteins from human skin is obtained, b) the mixture on the presence and amount of stratifin (14-3-3, ⁇ - Isoform), GRP78 / BiP, HSP27 (Heat shock protein 27 kDa), phosphorylated, in particular hyperphosphorylated isoforms of the HSP27 or fragments of these proteins and c) assigns the mixture examined in b) to old or stressed skin if it relates to the proteins listed in b) have a translation profile characteristic of old or stressed skin; or assigns the mixture of young or undressed skin examined in b) if it has a translation profile characteristic of young or undressed skin with regard to the proteins listed in b).
  • stratifin 14-3-3, ⁇ - Isoform
  • GRP78 / BiP H
  • a translation profile characteristic of old skin is present if the mixture contains either more different compounds typically translated in old skin than those that are typically expressed in young skin (qualitative differentiation), or contains more copies of compounds typically expressed in old skin, than typically present in young skin (quantitative differentiation).
  • the assignment to young or undressed skin is carried out in a complementary manner.
  • a characteristic translation profile is also present when several markers (translatio products of the genes which are important for skin aging and / or skin stress) are quantified, which must then be present in a characteristic relationship with one another in order to represent young skin, or in one of these different characteristic ratios must be present to represent old skin.
  • the mixture is preferably obtained from a skin sample, in particular from a whole skin sample or from an epidermis sample.
  • a skin sample opens up more extensive possibilities of comparison with the translation profiles, which are also related to whole skin or to corresponding in vitro or animal models.
  • the epidermis sample is easier to obtain, for example by applying an adhesive tape to the skin and tearing it off, as described in WO 00/10579, to which reference is hereby made in full.
  • the mixture is obtained in step a) by means of microdialysis.
  • microdialysis A method for measurement of local tissue metabolism", Nielsen PS, Winge K, Petersen LM; Ugeskr Laeger 1999 Mar 22 161: 12 1735-8; and in "Cutaneous microdialysis for human in vivo dermal absorption studies ", Anderson, C. et al. ; Drugs Pharm. 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 or fragments of proteins 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.
  • the method according to the invention for determining skin stress and / or skin aging is preferably carried out by examining the presence and amount of stratifin (14-3-3, ⁇ -isoform), GRP78 / BiP, HSP27 (Heat shock protein 27 kDa), phosphorylated, in particular hyperphosphorylated isoforms of the HSP27 or fragments of these proteins by separating the proteins by means of gel electrophoresis and subsequent Mass spectrometry, especially matrix assisted laser desorption ionization (MALDI).
  • stratifin 14-3-3, ⁇ -isoform
  • GRP78 / BiP Heat shock protein 27 kDa
  • HSP27 Heat shock protein 27 kDa
  • phosphorylated in particular hyperphosphorylated isoforms of the HSP27 or fragments of these proteins by separating the proteins by means of gel electrophoresis and subsequent Mass spectrometry, especially matrix assisted laser desorption ionization (MALDI).
  • MALDI matrix assisted laser desorption i
  • the separation of the proteins is particularly preferably carried out by 2D gel electrophoresis, as for example in L.D. Adams, Two-dimensional Gel E-lectrophoresis 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; Amer- sham Pharmacia Biotech, 1998 (order no. 80-6429-60).
  • the proteins are preferably obtained by protein precipitation, for example with a methanol / chloroform mixture in a ratio of about 4: 1: 4 to the sample and / or by concentrating the proteins, for example by ultrafiltration with a cut-off of about 5,000 Da, e.g. using the Vivaspin products from Sartorius.
  • the concentrate or precipitate thus obtained can then be taken up in rehydration buffer in a manner known to those skilled in the art, for example as in “2-D electrophoresis using immobilized pH gradients. Principles and meths ", Berkelman, T. and Stenstedt, T (eds); Amersham Pharmacia Biotech Ine (1998) 80-6429-60;
  • the separation of the proteins preferred according to the invention by 2-dimensional gel electrophoresis can be carried out, for example, by carrying out isoelectric focusing in the first dimension, for example using Immobilin DryStrips with an immobilized pH gradient (IPG) from 4 to 7 from Amersham -Pharmacia or using carrier ampholyte-carrying polyacrylamide gel strips which build up a pH gradient in the electric field (O'Farrell, PH; 1975; J Biol. Chem. 250, 4007-4021) and in the second dimension an SDS-polyacrylamide -Gelectrophoresis, e.g. with 12.5% polyacrylamide at 30 mA for 3 to 4 h.
  • Gel staining can be carried out by customary methods, for example by Heukeshoven silver staining or by colloidal Coomassie staining.
  • Another particularly preferred method for the detection of the above-mentioned proteins or protein fragments is the use of a protein chip.
  • Stratifin belongs to the large family of 14-3-3 proteins. Some members of this protein family are ubiquitously expressed (eg the ⁇ -isoform), while eg the ⁇ -isoform occurs specifically in T cells.
  • a form of the 14-3-3 protein produced specifically in stratified epithelia (eg in the epidermis) is the stratifin (the ⁇ -isoform).
  • An outstanding property of all 14-3-3 proteins is their ability to bind to different, functionally different signal proteins, such as kinases, phosphatases or transmembrane receptors.
  • An important biological function of stratifin was discovered when the growth of colonrectal carcinoma cells was inhibited with ⁇ -rays (Hermeking, H. et al., 1997, Mol. Cell. 1, 3-11).
  • the epidermis is a part of the skin that is constantly renewed. Divisible keratinocytes are located in the stratum basale, the deepest layer of the epidermis. These cells slowly migrate into higher layers of the epidermis, differentiating into the corneocytes of the stratum corneum. Stratifin can play a key role in this differentiation process.
  • amino acid sequence (A) is disclosed in the National Center for Biotechnology Information (NCBI) protein database under number 5454052.
  • the sequence of the corresponding mRNA is also disclosed there via a direct link. This database is accessible on the Internet at the following address: http://www.ncbi.nlm.nih.gov/.
  • stratifin also includes those proteins whose amino acid sequences can be obtained from the sequence (A) listed above by conservative mutations, that is to say by the exchange of structurally or functionally similar amino acids.
  • GRP's glucose-regulated proteins
  • HSP27 heat shock proteins
  • GRP78 / BiP belongs to these chaperones.
  • the binding of GRP78 / B ⁇ P e.g. of nascent proteins in the ER prevents the accumulation of denatured, i.e. unfolded, proteins.
  • GRP78 / BiP can dissociate from the bound proteins in an ATP-dependent reaction, so that they can then be folded correctly.
  • Another mechanism involves the targeted degradation of GRP78 / BiP-bound proteins via the ubiquitin / proteasome pathway.
  • HSP27 is also involved in the regulation of the cytoskeleton, in particular actin polymerization.
  • HSP27 human whole skin models with simulated sunlight also led to the induction of HSP27 synthesis in the applicant's experiments.
  • HSP-27 isoforms could be detected, which had different isoelectric points. These differences result from phosphorylation of the protein on serine residues that occur two amino acids after an arginine in the molecule.
  • hyperphosphorylated molecules could also be detected.
  • Phosphorylation of HSP27 like its synthesis, can be induced by the addition of the interleukin-1 cytokine. This cytokine is known (Wlaschek et al, 1997, FEBS Letters 413; 239-242) to be induced by UV light and to participate in the increased production of matrix metalloprotease-1 (a marker protein of photo-aged skin).
  • amino acid sequence (B) is disclosed in the National Center for Biotechnology Information (NCBI) protein database under number 6900104.
  • NCBI National Center for Biotechnology Information
  • the sequence of the corresponding mRNA is also available there via a direct link. beard. This database is accessible on the Internet at the following address: http://www.ncbi.nlm.nih.gov/.
  • GRP78 / BiP also includes those proteins whose amino acid sequences can be obtained from the sequence (B) listed above by conservative mutations, that is to say by the exchange of structurally or functionally similar amino acids.
  • HSP27 a protein with the amino acid sequence (C)
  • NCBI National Center for Biotechnology Information
  • HSP27 also includes those proteins whose amino acid sequences can be obtained from the above-mentioned sequence (C) by conservative mutations, that is to say by the exchange of structurally or functionally similar amino acids.
  • Another object of the present invention is a test kit for determining skin stress and / or skin aging in humans in vitro, comprising means for carrying out the method according to the invention for determining skin stress and / or skin aging.
  • Another object of the present invention is a biochip for determining skin stress and / or skin aging in humans in vitro, comprising i. a solid, ie rigid or flexible support and ii. on this immobilized probes that are capable of specific binding to at least one of the molecules selected from:
  • HSP27 Heat shock protein 27 kDa
  • 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, for example, also be formed 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 for years in Southern blot and Northern blot analysis. Compared to these conventional methods, in which only a few genes are analyzed, DNA chip technology allows a few hundred to several tens of thousands of genes to be examined in parallel.
  • a DNA chip essentially consists of a carrier material (e.g. glass or plastic) on which single-stranded, gene-specific probes are immobilized in a high density at a defined location (spot). The technique of probe application and the chemistry of probe immobilization are considered problematic.
  • EM 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 arrays by direct synthesis on a glass surface that was derivatized with 3-glycidoxypropyltrimethoxysilane and then with a glycol.
  • DNA molecules can also be bound to surfaces of support material.
  • P.O. Brown (DeRisi et al. (1997), Science 278, 680-686) describes the immobilization of DNA on glass surfaces coated with polylysine.
  • L.M. Smith (Guo, Z. et al. (1994), Nucleic Acid Research 22, 5456-5465) discloses a similar procedure: oligonucleotides bearing a 5'-terminal amino group can be bound to a glass surface which is coated with 3-aminopropyltrimethoxysilane and then treated with 1,4-phenyldiisothioeyanate.
  • 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: DNA probes are generated here by means of PCR using a sequence-specific pair of primers, with one primer at the 5 'end is modified and carries a linker with a free amino group. This ensures that a defined strand of the PCR products can be bound to a glass surface that has been treated with 3-aminopropyltrimethoxysilane and then with 1,4-phenyldiisothiocyanate.
  • the gene-specific PCR products should ideally have a defined nucleic acid sequence with a length of 200-400 bp and contain non-redundant sequences.
  • mRNA is isolated from two cell populations to be compared.
  • the isolated mRNAs are reverse transcribed using e.g. fluorescence-labeled nucleotides converted into cDNA.
  • the samples to be compared are e.g. marked with red or green fluorescent nucleotides.
  • the cDNAs are then hybridized with the gene probes immobilized on the DNA chip and the bound fluorescence is then quantified.
  • the biochip according to the invention preferably comprises 1 to approximately 5000, in particular 1 to approximately 1000, preferably approximately 10 to approximately 500, particularly preferably approximately 10 to approximately 250, particularly preferably approximately 10 to approximately 100 and very particularly preferably approximately 10 to approximately 50 from one another different probes.
  • the different probes can each be present in duplicate on the chip.
  • the biochip according to the invention preferably comprises nucleic acid probes, in particular RNA or PNA probes, particularly preferably DNA probes.
  • the nucleic acid probes preferably have a length of approximately 10 to approximately 1000, in particular approximately 10 to approximately 800, preferably approximately 100 to approximately 600, particularly preferably approximately 200 to approximately 400 nucleotides.
  • the biochip according to the invention comprises peptide or protein probes, in particular antibodies.
  • Another object of the present invention is the use of molecules which are selected from:
  • HSP27 Heat shock protein 27 kDa
  • Another object of the present invention is a test method for demonstrating the effectiveness of cosmetic or pharmaceutical active substances against skin stress and / or skin aging in vitro, characterized in that a) the skin status is determined by a method according to the invention for determining skin stress and / or skin aging, or using a test kit according to the invention for determining skin stress and / or skin aging, or using a biochip according to the invention, b) applying an active ingredient against skin stress and / or skin aging to the skin one or more times, c) renewing the skin status by means of an inventive method Method for determining skin stress and / or skin aging, or using a test kit according to the invention for determining skin stress and / or skin aging, or using a biochip according to the invention, and d) the effectiveness of the active ingredient by comparing the results from a) and c) determined.
  • Another object of the present invention is a test kit for demonstrating the effectiveness of cosmetic or pharmaceutical active substances against skin stress and / or skin aging in vitro, comprising means for carrying out the test method according to the invention.
  • Another object of the present invention is the use of molecules which are selected from:
  • HSP27 Heat shock protein 27 kDa
  • the present invention furthermore relates to a screening method for identifying cosmetic or pharmaceutical active substances against skin stress and / or skin aging in vitro, characterized in that a) the skin status is determined by a method according to the invention for determining skin stress and / or skin aging, or using a test kit according to the invention for determining skin stress and / or skin aging, or using a biochip according to the invention, b) applying a potential active substance against skin stress and / or skin aging to the skin one or more times, c) the skin status is determined again by a method according to the invention for determining skin stress and / or skin aging, or by means of a test kit according to the invention for determining skin stress and / or skin aging, or by means of a biochip according to the invention, and d) active ingredients by the Comparison of the results from a) and c) determined.
  • Another object of the present invention is the use of molecules which are selected from:
  • HSP27 Heat shock protein 27 kDa
  • Another object of the present invention is a method for producing a cosmetic or pharmaceutical preparation against skin stress and / or skin aging, characterized in that a) active ingredients with the aid of the screening method according to the invention, or the use for the identification of cosmetic or pharmaceutical active ingredients against skin stress and / or skin aging and b) active ingredients found to be effective mixed with cosmetically and pharmacologically suitable and compatible carriers.

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Abstract

L'invention concerne un procédé pour déterminer in vitro le stress et/ou le vieillissement de la peau chez l'homme, des kits de tests et des biopuces servant à la détermination du stress et/ou du vieillissement de la peau, et l'utilisation de protéines, de molécules mRNA ou bien de fragments de protéines ou de molécules mRNA comme marqueurs du stress et/ou du vieillissement de la peau. La présente invention porte également sur un procédé d'essai visant à prouver l'efficacité d'agents pharmaceutiques ou cosmétiques agissant contre le stress et/ou le vieillissement de la peau, sur un procédé de criblage pour identifier des agents pharmaceutiques ou cosmétiques agissant contre le stress et/ou le vieillissement de la peau, ainsi que sur un procédé pour produire une préparation pharmaceutique ou cosmétique agissant contre le stress et/ou le vieillissement de la peau.
PCT/EP2001/014812 2001-01-03 2001-12-14 Procede de determination in vitro du vieillissement de la peau WO2002054078A2 (fr)

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DE10100122.3 2001-01-03
DE10100122A DE10100122A1 (de) 2001-01-03 2001-01-03 Verfahren zur Bestimmung der Hautalterung in vitro

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WO2002054078A3 WO2002054078A3 (fr) 2003-02-27

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FR2924613A1 (fr) * 2007-12-10 2009-06-12 Oreal Utilisation cosmetique de proteines de type hsp27.
JP2012039970A (ja) * 2010-08-21 2012-03-01 Nippon Menaade Keshohin Kk 皮膚損傷の程度を検査する方法

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2847268B1 (fr) * 2002-11-19 2006-09-29 Coletica Procede d'identification d'une modification eventuelle d'au moins un parametre biologique mettant en oeuvre des cellules vivantes soumises a un stress et des cellules vivantes non soumises a ce meme stress
FR2847269B1 (fr) * 2002-11-19 2006-07-28 Coletica Procede d'identification d'une modification eventuelle d'au moins un parametre biologique mettant en oeuvre des cellules vivantes jeunes et agees

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