US20130184348A1 - Skin activation by acceleration of pdgf-bb activity - Google Patents

Skin activation by acceleration of pdgf-bb activity Download PDF

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US20130184348A1
US20130184348A1 US13/823,211 US201113823211A US2013184348A1 US 20130184348 A1 US20130184348 A1 US 20130184348A1 US 201113823211 A US201113823211 A US 201113823211A US 2013184348 A1 US2013184348 A1 US 2013184348A1
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pdgf
cells
skin
stem cells
mesenchymal stem
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Tsutomu Soma
Haruyo Yamanishi
Yumiko Ishimatsu
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Shiseido Co Ltd
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/67—Vitamins
    • A61K8/671—Vitamin A; Derivatives thereof, e.g. ester of vitamin A acid, ester of retinol, retinol, retinal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/18—Antioxidants, e.g. antiradicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5064—Endothelial cells
    • 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 skin activation by acceleration of platelet-derived growth factor-BB (PDGF-BB) activity.
  • PDGF-BB platelet-derived growth factor-BB
  • Stem cells are cells that have two properties consisting of pluripotency that allows production of cells that differentiate into a plurality of cells, and self-replication that allows production of cells that are identical to those cells.
  • Stem cells derived from embryos which are the initial development stage of a fertilized egg, are referred to as embryonic stem cells (ES cells).
  • ES cells embryonic stem cells
  • human ES cells are expected to be used in regenerative medicine, the production of new human ES cells is not allowed due to ethical considerations involving the use of fertilized eggs.
  • iPS cells induced pluripotent stem cells
  • somatic stem cells which have the ability to differentiate into specific tissue, are not associated with ethical issues in the manner of embryonic stem cells since they are obtained from the patient's own bone marrow or other body tissue.
  • Non-Patent Document 1 epidermal stem cells
  • Non-Patent Document 2 follicular epithelial stem cells
  • Non-Patent Document 3 melanocyte stem cells
  • fibroblasts having a long, narrow spindle shape are present in fibrous components of the skin consisting mainly of collagen, it has not yet been determined as to whether stem cells are present in dermal fibroblasts.
  • mesenchymal stem cells which are isolated from bone marrow as fibroblast precursors (Non-Patent Document 5), differentiate into various cells belonging to mesenchymal cell lines (including osteocytes, myocytes, chondrocytes, tendon cells and adipocytes), they are expected to be applied to regenerative medicine such as the reconstruction of bone, blood vessels and muscle. More recently, mesenchymal stem cells have been determined to have the potential of being present in numerous tissues having mesenchymal tissue, and have been isolated from fat, umbilical cord blood and the placenta (Non-Patent Documents 6 to 8). However, the presence of mesenchymal stem cells in the dermis has yet to be determined.
  • Non-Patent Document 1 Watt, F. M., J. Dermatol. Sci., 28: 173-180, 2002
  • Non-Patent Document 2 Cotsarelis, G., et al., Cell, 57: 201-209, 1989
  • Non-Patent Document 3 Nishimura, E. K., et al., Nature, 416: 854-860, 2002
  • Non-Patent Document 4 Wong, C. E., et al., J. Cell Biol., 175: 1005-1015, 2006
  • Non-Patent Document 5 Pittenger, M. F., et al., Science, 284: 143-147, 1999
  • Non-Patent Document 6 Park, K. W., et al., Cell Metab., 8: 454-457, 2008
  • Non-Patent Document 7 Flynn, A., et al., Cytotherapy, 9: 717-726, 2007
  • Non-Patent Document 8 Igura, K., et al., Cytotherapy, 6: 543-553, 2004
  • Non-Patent Document 9 Kim, W. S., et al., J. Dermatol. Sci., 53: 96-102, 2009
  • Non-Patent Document 10 Dalla-Favera, R., et al., Nature, 292: 31-35, 1981
  • Mesenchymal stem cells have been determined to also be present in fat in addition to bone marrow, umbilical cord blood and the placenta. Mesenchymal stem cells have also been determined to be present in the dermis in the same manner as subcutaneous fat located below the dermis, and further determined that they locally present at vascular sites. However, the mechanism by which these mesenchymal stem cells of the dermis and fat are locally present at vascular sites, as well as the reason why these stem cells increase or decrease with aging, are unknown.
  • an object of the present invention is to determine the reason for increases or decreases in mesenchymal stem cells in the dermis and subcutaneous fat caused by aging, and provide a method for improving skin condition by regulating those factors involved in maintaining mesenchymal stem cells at these sites.
  • mesenchymal stem cells are also present in the dermis in the same manner as subcutaneous fat, and established a method for isolating these mesenchymal stem cells present in the dermis and subcutaneous fat (Japanese Patent Application No. 2009-213921).
  • Non-Patent Document 9 Fat-derived mesenchymal stem cells have been determined to demonstrate an antioxidant function in the skin.
  • skin aging can be inhibited by enhancing the expression of endogenous PDGF-BB at vascular sites where dermal and subcutaneous fat-derived mesenchymal stem cells are present locally, and more specifically, in vascular endothelial cells that highly express PDGF-BB, and maintaining a large number of mesenchymal stem cells in the dermis and subcutaneous fat.
  • [3] a method for inhibiting skin aging by activating mesenchymal stem cells, and thereby activating skin, by increasing the activity or level of PDGF-BB at a vascular site of the skin;
  • a novel skin activator can be identified by the present invention.
  • FIG. 1 indicates expression of PDGF gene in vascular endothelial cells.
  • FIG. 2 indicates migration of dermal/adipose-derived stem cells induced by PDGF.
  • FIG. 3 indicates localization of PDGF-BB in dermis.
  • FIG. 4 indicates localization of PDGF-BB in dermis.
  • FIG. 5 indicates the effects of PDGF-BB inhibition on accumulation of dermal/adipose-derived stem cells in niches.
  • FIG. 6 indicates changes in human dermal/adipose-derived stem cells attributable to aging.
  • FIG. 7 indicates changes in human dermal/adipose-derived stem cells attributable to aging.
  • FIG. 8 indicates acceleration of PDGF-BB production by retinoic acid.
  • the present invention relates to a method for screening drugs that activate skin, comprising allowing a candidate drug to act on vascular endothelial cells, and selecting the drug that accelerates expression of PDGF-BB by the cells for use as a skin activator.
  • Platelet-derived growth factor is a growth factor involved in regulation of migration, proliferation or the like of fibroblast, smooth muscle cells, glial cells and other mesenchymal cells, and is produced by various cells such as epithelial cells or endothelial cells.
  • PDGF a growth factor involved in regulation of migration, proliferation or the like of fibroblast, smooth muscle cells, glial cells and other mesenchymal cells
  • various cells such as epithelial cells or endothelial cells.
  • PDGF-A, PDGF-B, PDGF-C and PDGF-D there are also three types of isoforms (PDGF-AA, PDGF-AB and PDGF-BB) resulting from the adoption of a homo- or hetero-dimer structure as a result of the A chain and B chain forming disulfide bonds.
  • PDGF is known to express its physiological action through PDGF receptor (PDGFR) which is a tyrosine kinase-associated receptor.
  • PDGFR PDGF receptor
  • PDGF-B gene is known and has been cloned (Non-Patent Document 10).
  • the mesenchymal stem cells used in the present invention can be derived from the dermis of all species of mammals, including humans, chimpanzees, other primates, domestic animals such as dogs, cats, rabbits, horses, sheep, goats, cows and pigs, as well as laboratory animals such as rats, mice and guinea pigs.
  • activation of skin generally refers to a state in which metabolism of skin tissue becomes active, the turnover period becomes comparatively short, and tissue fatigue, atrophy and the progression of oxidation are reduced. As a result of tissue activation, skin tightness can be maintained and the formation of wrinkles and age spots can be prevented and alleviated.
  • Expression of PDGF-BB gene in vascular endothelial cells may be determined by, for example, measuring the level of PDGF-BB.
  • this measurement can be carried out by a method commonly known in the relevant technical field using an antibody specific to PDGF-BB, examples of which include various methods such as immunostaining using a fluorescent substance, pigment, enzyme or the like, Western blotting or immunoassay such as ELISA or RIA.
  • this can also be determined by extracting total RNA from vascular endothelial cells, and measuring the amount of mRNA that encodes PDGF-B.
  • RNA is extracted from RNA and measurement of the amount thereof.
  • quantification of RNA is carried out by quantitative polymerase chain reaction (PCR) such as real-time polymerase chain reaction (RT-PCR).
  • PCR quantitative polymerase chain reaction
  • RT-PCR real-time polymerase chain reaction
  • Selection of primers suitable for RT-PCR can be carried out by a method commonly known among persons with ordinary skill in the art.
  • retinoic acid accelerates expression of PDGF-BB.
  • selection of a skin activator can be carried out by investigating by means of statistical techniques and the like whether or not a candidate drug accelerates expression of PDGF-BB by comparing with a positive control, which uses a drug such as retinoic acid having an effect of accelerating expression of PDGF-BB, and a negative control, which uses a drug such as siRNA of PDGF-B gene having an effect of inhibiting expression of PDGF-BB.
  • the present invention also provides a method for evaluating a candidate drug for the ability to accelerate expression of a polynucleotide capable of hybridizing under highly stringent conditions with a polynucleotide that encodes PDGF-B (SEQ ID NO: 1) in vascular endothelial cells, and selecting a candidate drug that has that accelerating ability for use as a skin activator.
  • Hybridization can be carried out in accordance with a commonly known method or method applicable thereto, such as the method described by Sambrook, et al. in Molecular Cloning 2nd Edition, Cold Spring Harbor Lab.
  • highly stringent hybridization conditions refer to conditions consisting of, for example, a sodium concentration of about 10 mM to 40 mM and preferably about 20 mM, and a temperature of about 50° C. to 70° C. and preferably about 60° C. to 65° C.
  • the present invention also relates to an aesthetic method for activating skin and thereby inhibiting skin by increasing the activity or level of PDGF-BB at vascular sites of skin to allow PDGF-BB, for which activity has been accelerated or expression level has been increased, to act on mesenchymal stem cells and activate the mesenchymal stem cells as a result thereof.
  • retinoic acid and particularly tretinoic acid, accelerates expression of PDGF-BB.
  • this method is carried out by applying tretinoic acid to, for example, skin for which aging is desired to be inhibited.
  • the activity or level of PDGF-BB can be increased by using a vector incorporating PDGF-BB gene in order to introduce the PDGF-B gene per se into cells.
  • a regulatory sequence such as a promoter or enhancer that accelerates expression of PDGF-B gene is preferably arranged at a location that enables it to act on the PDGF-B gene.
  • Either a gene insertion method using a viral vector or a non-viral gene insertion method can be applied for introducing the PDGF-B gene into cells.
  • Examples of gene insertion methods using a viral vector include methods consisting of incorporating DNA encoding PDGF-B and inserting into a DNA or an RNA virus such as a retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, pox virus, polio virus or sinbis virus.
  • non-viral gene insertion methods include a method consisting of intramuscular administration of an expression plasmid (DNA vaccine method), liposome method, lipofectin method, microinjection method, calcium phosphate method and electroporation, and the DNA vaccine method and liposome method are particularly preferable.
  • DNA vaccine method intramuscular administration of an expression plasmid
  • liposome method lipofectin method
  • microinjection method calcium phosphate method and electroporation
  • the DNA vaccine method and liposome method are particularly preferable.
  • ex vivo methods in which a certain type of cells are extracted from a human, DNA is introduced into the cells outside the body, and the cells are then returned to the body (Nikkei Science, April 1994, pp.
  • In vivo methods are more preferable.
  • administration can be made, for example, intravenously, intraarterially, subcutaneously, intracutaneously or intramuscularly.
  • administration is typically made in the form of an injection preparation and the like, and a commonly used vehicle may be added as necessary.
  • administration in the form of liposomes or fusogenic liposomes (such as Sendai virus-liposome fusion products), administration can be in the form of a liposome preparation such as a suspension, frozen preparation or centrifugally separated frozen concentrate.
  • a liposome preparation such as a suspension, frozen preparation or centrifugally separated frozen concentrate.
  • the expression level of PDGF-B gene in constituent cells of human skin was investigated by quantitative PCR.
  • Epidermal keratinocytes KC and follicular epithelial cells in the form of hair follicle outer root sheath cells ORSC were cultured using EpiLife-KG2 medium (Kurabo Industries Ltd.)
  • skin fibroblasts FB were cultured using DMEM medium containing 10% FBS (Invitrogen Corp.)
  • human vascular endothelial cells HUVEC were cultured using EGM-2 medium (Sanko Junyaku Co., Ltd.).
  • Isogen Nippon Gene Co., Ltd.
  • total RNA was extracted in accordance with the protocol provided.
  • RNA Concentration of the purified total RNA was measured with the Nanodrop nucleic acid quantification analyzer (Thermo Scientific Inc.). Each sample was then used to synthesize cDNA in accordance with the manual provided by Invitrogen Corp. using random primers (Invitrogen Corp.) and Superscript II reverse transcriptase (Invitrogen Corp.). Quantitative PCR was then carried out by using the synthesized cDNA as a template and using LightCycler FastStart DNA Master PLUS SYBR Green (Roche Diagnostics GmbH) for the reaction reagent and LightCycler (Roche Diagnostics GmbH) for the reaction device. Compositional conditions were in accordance with the Roche protocol. In addition, PCR conditions consisted of initial denaturation for 10 minutes at 95° C.
  • PDGF-A (SEQ ID NO: 1) Forward primer: 5′-ATACCTCGCCCATGTTCTG-3′ (SEQ ID NO: 2) Reverse primer: 5′-GATGCTTCTCTTCCTCCGAA-3′ PDGF-B: (SEQ ID NO: 3) Forward primer: 5′-CTTTAAGAAGGCCACGGTGA-3′ (SEQ ID NO: 4) Reverse primer:5′-CTTCAGTGCCGTCTTGTCAT-3′ PDGF-C: (SEQ ID NO: 5) Forward primer: 5′-TATATTAGGGCGCTGGTGTG-3′ (SEQ ID NO: 6) Reverse primer: 5′-ATTAAGGAGGTCCAGTGGCA-3′ PDGF-D: (SEQ ID NO: 7) Forward primer: 5′-TGGGAATCTGTCACAAGCTC-3′ (SEQ ID NO: 8) Reverse primer: 5′-CTTTTGACTTCCGGTCATGG-3′ G3PDH: (SEQ ID NO: 9) Forward primer: 5′-GCACCGTC
  • G3PDH was used as an internal standard, and this was used to correct cDNA of the control group during quantification of each gene.
  • adipose-derived mesenchymal stem cells MSC were purchased and sub-cultured in MesenPro mesenchymal stem cell medium (Invitrogen Corp.). Then, PDGF-AA, PDGF-AB or PDGF-BB (R&D Systems Inc.) was added to StemPro serum-free MSC medium (Invitrogen Corp.) in 24-well culture plates at a concentration of 5 ng/ml to 30 ng/ml, and fibronectin-coated cell inserts (BD Bioscience Inc.) were placed thereon followed by seeding 50,000 MSC suspended in StemPro medium. After culturing overnight in a CO 2 incubator, the culture fluid was removed by aspiration.
  • MesenPro mesenchymal stem cell medium Invitrogen Corp.
  • PDGF-AA, PDGF-AB or PDGF-BB R&D Systems Inc.
  • StemPro serum-free MSC medium Invitrogen Corp.
  • fibronectin-coated cell inserts BD Bioscience Inc
  • the cell inserts were immersed for 10 minutes in Hoechist 33258-PBS solution, and the nuclei of cells adhered to the cell insert were stained. After washing with PBS, the backs of the cell inserts were observed under a fluorescence microscope and images were captured. Five random images of each cell insert were captured followed by counting the number of cells that transferred to the cell insert.
  • the sections were washed a total of three times consisting of twice for 40 minutes each with TBST and once for 40 minutes with TBS, and after nuclear staining with Hoechist 33258, the sections were observed and imaged using an LSM5 PASCAL confocal fluorescence microscope (Zeiss GmbH).
  • HUVEC labeled with red fluorescent pigment PSH26 Red Fluorescent, Sigma Corp.
  • MSC labeled with green fluorescent pigment PH67 Green Fluorescent, Sigma Corp.
  • the slide was incubated for 12 hours at 37° C. in the presence of 5% CO 2 .
  • the status of the tubes that formed was observed using an LSM5 PASCAL confocal fluorescence microscope (Zeiss GmbH) and images were captured.
  • mouse anti-PDGF receptor neutralizing antibody R&D Systems Inc.
  • mouse IgG coinciding with the isotype were used at a concentration of 5 ⁇ g/ml.
  • PDGF-B (SEQ ID NO: 11) Forward primer: 5′-CCTGGCATGCAAGTGTGA-3′ (SEQ ID NO: 12) Reverse primer: 5′-CCAATGGTCACCCGATTT-3′
  • tissue After fixing human skin tissue for up to 1 week in formalin-phosphate buffer solution, the tissue was embedded in paraffin using an automated embedder (Sakura Finetek Japan Co., Ltd.).
  • Six ⁇ m tissue sections were prepared from the resulting human skin paraffin block with a microtome (Leica Microsystems GmbH), and the sections were affixed to an APS coated slide glass followed by flattening and drying on a flattening table (Sakura Finetek Japan Co., Ltd.).
  • the prepared skin tissue slides were de-paraffinized with xylene and subjected to hydrophilic treatment with a mixture of ethanol and water, and after rinsing with TBS buffer, CD34 antigen was activated by subjecting to enzymatic reaction treatment at 37° C.
  • Human vascular endothelial cells HUVEC were sub-cultured in EGM-2 medium (Sanko Junyaku Co., Ltd.), and the fourth generation cells were suspended in Humedia-EG2 medium not containing VEGF-A (Kurabo Industries Ltd.), followed by disseminating in a collage-coated 24-well multiplate (Asahi Glass Co., Ltd.) at a ratio of 20,000 cells and culturing for 3 to 5 days at 37° C. until the cells reached confluence in the presence of 5% CO 2 .
  • culturing was further continued for 2 days.
  • the culture supernatant was recovered and PDGF-BB was quantified in accordance with the protocol provided using Human PDGF-BB Quantikine ELISA Kit (R&D Systems Inc.).
  • mRNA was extracted and purified from the cultured cells using the RNA extraction reagent, MagNA Pure LC mRNA HS Kit (Roche Diagnostics GmbH), and the automated nucleic acid extraction system, MagNA Pure LC 1.0 Instrument (Roche Diagnostics GmbH) in accordance with the protocol provided.
  • RT-PCR was then carried out on the PDGF-B gene for each sample using an equal volume of mRNA as template, using the primer pair of SEQ ID NO: 9 and SEQ ID NO: 10, and using QuantiFast SYBR Green RT-PCR Kit (Qiagen Inc.) for the reaction reagent and LightCycler (Roche Diagnostics GmbH) for the reaction apparatus. Compositional conditions were in accordance with the Qiagen protocol. In addition, RT-PCR conditions consisted of carrying out the RT reaction for 20 minutes at 50° C., initial denaturation for 15 minutes at 95° C., denaturation for 15 seconds at 94° C., annealing for 20 seconds at 60° C., and extension for 30 seconds at 72° C. Furthermore, G3PDH was used as an internal standard, and this was used to correct the amount of mRNA of the control group.
  • Mesenchymal stem cells have been determined to be locally present at vascular sites in human skin (Japanese Patent Application No. 2009-213291).
  • PDGF-A, PDGF-B, PDGF-C and PDGF-D were determined to be extremely high expressed in HUVEC while hardly expressed at all in FB ( FIG. 1 ).
  • PDGF-A was determined to be expressed equally in KC, ORSC and HUVEC and at about four times the level of that in FB, while PDGF-C and PDGF-D were expressed at considerably high levels in FB ( FIG. 1 ). Since expression of PDGF-A and PDGF-B was observed in HUVEC, it was decided to investigate the effects of PDGF proteins PDGF-AA, PDGF-AB and PDGF-BB arising from these genes on the migration ability of mesenchymal stem cells. As a result, PDGF-BB was determined significantly enhance the migration ability of stem cells in comparison with PDGF-AA and PDGF-AB ( FIG. 2 ).
  • tretinoin all-trans retinoic acid

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JP5933443B2 (ja) 2016-06-08
TW201216973A (en) 2012-05-01
US20160002724A1 (en) 2016-01-07
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US10017817B2 (en) 2018-07-10
JPWO2012036211A1 (ja) 2014-02-03

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