WO2007102572A1 - Inhibiteur de vieillissement cellulaire - Google Patents

Inhibiteur de vieillissement cellulaire Download PDF

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WO2007102572A1
WO2007102572A1 PCT/JP2007/054513 JP2007054513W WO2007102572A1 WO 2007102572 A1 WO2007102572 A1 WO 2007102572A1 JP 2007054513 W JP2007054513 W JP 2007054513W WO 2007102572 A1 WO2007102572 A1 WO 2007102572A1
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protein
subunit
gene
gng11
cell
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PCT/JP2007/054513
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Japanese (ja)
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Dai Ayusawa
Mohammad Nazir Hossain
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Yokohama City University
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Publication of WO2007102572A1 publication Critical patent/WO2007102572A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/13—Nucleic acids or derivatives thereof
    • 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
    • A61K8/60—Sugars; Derivatives thereof
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it

Definitions

  • the present invention relates to a cell aging inhibitor that suppresses cellular senescence.
  • Non-Patent Document 1 Hayflick, L., Moorehead P.S.The serial cultivation of human diploid cell strains. Exp cell Res 25, 585—621 (1961).
  • Non-Patent Document 2 Herbig, U. & Sedivy, J. M. Regulation of growth arrest in senescence: Telomere damage is not the end of the story.Mech Ageing Dev (2005).
  • Non-Patent Document 3 Lin, A. W. et al. Premature senescence involving p53 and pl6 is activ atea in response to constitutive MEK / MAPK mitogenic signaling. Genes Dev 12, 30 08-19 (1998).
  • Non-Patent Document 4 Wang, W. et al. Sequential activation of the MEK-external signal —regulated kinase and MKK3 / 6-p38 mitogen— activated protein kinase pathways med iates oncogenic ras- induced premature senescence. Mol Cell Biol 22, 3389-403 (20 02).
  • Non-Special Terms 5 Sumikawa, ⁇ ⁇ , Matsumoto, ⁇ ⁇ , Sakemura, R., Fujii, M. & Ayusawa, D. Prolonged unbalanced growth induces cellular senescence markers linked with mech ano transduction in normal and tumor cells. Biochem Biophys Res Commun 335, 558 -65 (2005).
  • Non-Patent Literature 6 Jakab, M. et al. Mechanisms sensing and modulating signals arising fr om cell swelling. Cell Physiol Biochem 12, 235-58 (2002).
  • Non-Patent Document 7 Samarakoon, R. & Higgins, PJ MEK / ERK pathway mediates cell-sh ape-dependent plasminogen activator inhioitor type 1 gene expression upon drug-in prised disruption of the microfilament and microtubule networks. J Cell Sci 115, 309 3-103 (2002).
  • Non-Special Reference 8 Harman, D. Prolongation of the normal life span by radiation protecti on chemicals. J Gerontol 12, 257 ⁇ D3 (1957).
  • Non-Patent Document 9 Suzuki, T. et al. Induction of senescence-associated genes by 5-brom odeoxyuridine in HeLa cells. Exp Gerontol 36, 465-74 (2001).
  • Non-Special Terms 10 Minagawa, S., Fujii, M., Scherer, S. W. & Ayusawa, D. Functional a nd chromosomal clustering of genes responsive to 5— bromodeoxyuridine in human ce lis. Exp Gerontol in press (2004).
  • Non-Special Reference 11 Michishita, E. et al. 5-Bromodeoxyuridine induces senescence-like phenomena in mammalian cells regardless of cell type or species.J Biochem (Tokyo) 126, 1052-9 (1999).
  • Non-Special Reference 12 Gilman, A. G. G proteins: transducers of receptor-generated signals. Annu Rev Biochem 56, 615-49 (1987).
  • Non-Patent Document 13 Simon, M. I., Strathmann, M. P. & Gautam, N. Diversity of G protei ns in signal transduction, science 252, 802-8 (1991).
  • Non-Special Reference 14 Birnbaumer, L. Receptor-to-effector signaling through G proteins: roles for beta gamma dimers as well as alpha subunits.Cell 71, 1069-72 (1992).
  • Non-Special Terms 15 Wickman, KD et al. Recombinant G-protein beta gamma-subunits activate the muscarinic-gated atrial potassium channel. Nature 368, 255-7 (1994).
  • Non-Special Terms lb Clapham, DE & Neer , EJ New roles for G-protein beta gamma-dimers in transmembrane signaling. Nature 365, 403-6 (1993).
  • Non-Special Reference 17 Pitcher, J. A. et al. Role of beta gamma subunits of G proteins in tar geting the beta-adrenergic receptor kinase to membrane-bound receptors. Science 257, 1264-7 (1992).
  • Non-Special Publication 18 Haga, K. & Haga, T. Activation by G protein beta gamma subunits of f agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptors and rhodopsin.J Biol Chem 267, 2222-7 (1992).
  • Non-Special Reference 19 Balcueva, E.A. et al. Human G protein gamma (ll) and gamma (14) s ubtypes define a new functional subclass.Exp Cell Res 257, 310-9 (2000).
  • Non-Patent Document 20 Ray, ⁇ ⁇ , Kunsch, C "Bonner, LM & Robishaw, JD Isolation of c DNA clones encoding eight different human G protein gamma subunits, including thr ee novel forms designated the gamma 4, gamma 10, and gamma ⁇ ⁇ subunits. J Biol C hem 270, 21765-71 (1995).
  • Non-Patent Document 21 Lim, W. ⁇ ⁇ , Myung, CS, Garrison, JC & Neubig, RR Receptor-G protein gamma specificity: gamma 11 shows unique potency for A (l) adenosine an d 5— HT (1A) receptors Biochemistry 40, 10532-41 (2001).
  • Patent Document 22 Ide, A., Fujii, Tsuji, Nakababashi, K. & Ayusawa, D. Suppression of se inescence in normal human fibroblasts by introduction of dominant-negative p53 mut ants or human papilloma virus type 16 E6 protein. Biosci Biotechnol Biochem 62, 14 58-60 (1998).
  • Non-Special Terms 23 Brown, J. P., Wei, W. & Sedivy, J. M. Bypass of senescence after di sruption of p21CIPl / WAFl gene in normal diploid human fibroblasts, science 277, 8 31-4 (1997).
  • Non-Patent Document 24 Wei, W “Herbig, U” Wei, S “Dutriaux, A. & Sedivy, JM Loss of r etinoolastoma but not pl6 function allows bypass of replicative senescence in human fibroblasts. EMBO Rep 4, 1061—6 (2003).
  • Non-Patent Document 25 Chen, QM et al. Molecular analysis of H202- induced senescent- like growth arrest in normal human fibroblasts: p53 and Rb control Gl arrest but not cell replication.Biochem J 332 (Pt 1), 43-50 (1998).
  • Non-Patent Document 26 DeYulia, G. J "Jr., Carcamo, J. M., Borquez- Ojeda, O” Shelton, C.
  • Patent Literature 27 Mukmn, YV et al. 5-Hydroxytryptamine 1 A receptor / Gibetagamm a stimulates mitogen- activated protein kinase via NAD (P) H oxidase and reactive ox ygen species upstream of src in Chinese hamster ovary fibroblasts.Biochem J 347 Pt
  • Non-Patent Document 28 Matsukawa, J., Matsuzawa, A., Takeda, K. & Ichijo, H. The ASK1- MAP kinase cascades in mammalian stress response.J Biochem (Tokyo) 136, 2ol-5 (2004).
  • An object of the present invention is to provide a novel cell aging inhibitor capable of effectively and fundamentally suppressing cell aging.
  • the inventors of the present application have found various genes as a result of various searches by gene array analysis for genes whose expression increases in aging cells. Of these, we focused on the gene for sputum subunit 11 of the G protein.
  • Antisense cDNA for G protein ⁇ subunit 11 gene (3) The G protein ⁇ subunit 11 gene is induced very rapidly by an aging inducer such as hydrogen peroxide.
  • an aging inducer such as hydrogen peroxide.
  • the present invention relates to a cell aging inhibitor comprising, as an active ingredient, a substance that inhibits the expression of a gene encoding G protein ⁇ subunit 11 or a substance that inhibits the cellular aging action of G protein ⁇ subunit 11 I will provide a.
  • the present invention also relates to the use of a substance that inhibits the expression of a gene encoding G protein ⁇ subunit 11 or a substance that inhibits the cellular senescence action of G protein ⁇ subunit 11 for the production of a cell aging inhibitor. provide.
  • the present invention provides a cell comprising administering to an individual an effective amount of a substance that inhibits expression of a gene encoding G protein ⁇ subunit 11 or a substance that inhibits the cellular senescence action of G protein ⁇ subunit 11.
  • a cell comprising administering to an individual an effective amount of a substance that inhibits expression of a gene encoding G protein ⁇ subunit 11 or a substance that inhibits the cellular senescence action of G protein ⁇ subunit 11.
  • the present invention provides a novel cell aging inhibitor capable of effectively and fundamentally suppressing cell aging.
  • the cell aging inhibitor of the present invention induction of cell aging due to oxidative stress can be prevented, and thus various applications based on cell rejuvenation are possible. In particular, it can be applied to the development of cosmetics, health foods, and pharmaceuticals that are effective in preventing aging.
  • FIG. 1 is a graph showing an increase in the expression of GNG-11 gene in aged normal human fibroblast TIG-7.
  • Young human normal fibroblasts TIG-7 32 PDL
  • resting cells 5-day serum starvation
  • senescent cells 75 PDL
  • Total mRNA samples were prepared and GN G-ll mRNA levels were quantified by Northern blot analysis.
  • FIG. 2 is a diagram showing the induction of GNG-11 gene expression by hydrogen peroxide.
  • Total mRNA samples were prepared by adding peroxyhydrogen to the culture medium of young human normal fibroblasts TIG-7 (42PDL). Changes in GNG-llmRNA levels were quantified by Northern blot analysis.
  • FIG. 3 is a diagram showing the induction of cell senescence marker gene expression.
  • a GNG-11 expression vector was introduced into a normal human fibroblast (32PDL), and a Northern blot analysis was performed using a total mRNA sample prepared after 3 days of culture.
  • FIG. 4 is a diagram showing prolongation of mitotic life by introduction of antisense cDNA.
  • An antisense GNG-11 cDNA expression vector was introduced into the immediately preceding normal human fibroblast TIG-7 (PDL62), and the transfected cells were selected with G418 selection medium. Stain colonies and count the number did.
  • FIG. 5 shows changes in mitotic life due to introduction of a GNG-11 expression plasmid.
  • a sense GNG-11 cDNA or an antisense GNG-11 cDNA expression vector was introduced into young normal fibroblast TIG-7 (PDL32, arrow), and the gene-transferred cells were selected using a G418 selective medium. Colonies were isolated and their mitotic life was measured.
  • G protein ⁇ subunit 11 belongs to the ⁇ subunit family of G proteins (ten or more are known). G protein is composed of ⁇ , ⁇ , and ⁇ ⁇ subunits. When a ligand binds to a cell membrane receptor, the G protein is activated and dissociates into ⁇ and
  • the Ras protein which has the potential to function as a shunt for signaling transmission, has recently been increasingly involved in cellular senescence.
  • the base sequence of GNG11 gene and the amino acid sequence of GNG11 encoded by it are known.
  • the base sequence of cDNA of human GNG11 gene and the amino acid sequence of GNG11 encoded by it are described in GenBank Accession No. NM_004 126 (SEQ ID NOS: 1 and 2 in the Sequence Listing).
  • the degree of aging of cells is determined by morphological observation of cells (when cells aging, flattening and hypertrophy) and ⁇ -galactosidase staining image known as an aging marker (when aging progresses, ⁇ -galatatosidase staining) It can be examined by observing (the image becomes larger).
  • the cell aging inhibitor of the present invention contains a substance that inhibits the expression of the GNG11 gene or a substance that inhibits the cell aging action of GNG11 as an active ingredient.
  • substances that inhibit GNG11 gene expression include antisense nucleic acids, interfering RNAs, peptides, and plant components for the GNG11 gene.
  • an antisense nucleic acid can be easily prepared based on a conventional method.
  • the antisense nucleic acid may be an antisense RNA or an antisense cDNA that generates antisense RNA by transcription in a cell.
  • Antisense RNA against the GNG11 gene is RNA that hybridizes with mRNA transcribed from the GNG11 gene and inhibits translation of the mRNA.
  • Antisense RNA is most preferably complementary to GNG11 mRNA. However, if there is a degree of complementarity that can hybridize with mRNA in the cell, use one with a slight mismatch.
  • the nucleotide sequence identity of the antisense RNA to the complementary strand of GNG11 mRNA is preferably 90% or more, and more preferably 95% or more.
  • the identity of the base sequence is determined by aligning the sequences so that the number of matching bases is the largest, and by matching the number of bases that match the total number of bases (if the total number of bases is different Is the percentage divided by the number of short bases) and can be easily calculated by known software such as BLAST.
  • the size of the antisense RNA is preferably at least 70% of the total length of the GNG11 cDNA, or a size that can specifically hybridize with the mRNA of GNG11, preferably 18 bases or more. Is available.
  • an antisense RNA that hybridizes to a region including the entire length of the coding region or a region within the coding region is preferred! /.
  • a recombinant vector in which an antisense cDNA is incorporated and the antisense cDNA is transcribed in the cell to produce the antisense RNA can be used (see below). See Examples). Since expression vectors for mammals are well known and various types are commercially available, commercially available expression vectors can be used.
  • various plasmid vectors and virus vectors that are used as vectors for gene therapy and gene vaccines can be used.
  • Recombinant vectors in which DNAs that produce antisense RNA against the GNG11 gene described above by transcription are incorporated into these commercially available expression vectors can be preferably used as the active ingredient of the cell aging inhibitor of the present invention.
  • RNAi RNA interference method
  • siRNA double-stranded short RNA
  • the siRNA may be a double-stranded RNA having the same base sequence as an arbitrary partial region of the GNG11 cDNA (SEQ ID NO: 1), but the mRNA translation can be surely inhibited by mRNA cleavage.
  • interfering RNA having the same nucleotide sequence as the region within the coding region of 352nt ⁇ 573 n t is preferred.
  • a substance that inhibits the cell aging action of GNG11 itself should also be used as an active ingredient of the cell aging inhibitor of the present invention. Can do.
  • an antibody against GNG11 can be mentioned.
  • the antibody may be a monoclonal antibody or a polyclonal antibody, but a polyclonal antibody includes antibodies against all exposed GNG11 epitopes, and thus can reliably inhibit the cellular senescence action of GNG11.
  • GNG11 specifically binds to other subunits of G protein, the binding domain of G protein ⁇ or ⁇ subunit can be used in the same manner as an antibody. When GN G11 binds to the binding domain of the G protein or ⁇ subunit, it can no longer bind to the a or j8 subunit of the G protein. Is also considered to be inhibited.
  • the cell aging inhibitor of the present invention is used as a pharmaceutical and as a component of cosmetics and health foods.
  • RNA or interfering RNA against GNG11 gene a solution obtained by dissolving antisense RNA or interfering RNA in a medium such as physiological buffer saline is directly injected into a tissue in which suppression of cell aging is desired. be able to. It can also be introduced into the organization by the electric pulse method or ultrasonic method.
  • the dosage in this case is appropriately selected according to the symptoms and the like, but is usually about antisense RNA or interfering RNA lpmol to l nmol per lg of target tissue.
  • a solution obtained by dissolving the recombinant vector in a medium such as physiological buffer saline can be directly applied to tissues in which inhibition of cell aging is desired, intramuscularly, or subcutaneously. Can be injected.
  • the dose in this case is appropriately selected according to the symptoms and the like, but it is usually about 1 pmol to l nmol of recombinant vector per lg of target tissue.
  • the active ingredient is an anti-GNG11 antibody
  • the dose is appropriately selected according to the symptom and the like, but is usually about 1 ⁇ g to 100 ⁇ g of antibody per lg of target tissue.
  • Active ingredients such as antisense RNA, recombinant vector incorporating antisense cDNA, and anti-GNG11 antibody are administered systemically by parenteral or oral administration such as intravenous administration, intramuscular administration, or subcutaneous administration. It is also possible.
  • the dose in the case of whole body administration is appropriately selected according to the symptom etc. Usually, however, the dose per lg body weight should be approximately the same as 1/1000 of the dose in the case of direct injection described above. The amount is sufficient.
  • antisense RNA or antibody it can be applied to the skin as a cosmetic ingredient. In this case, the content in the cosmetic is appropriately selected, but may be about 1 g to 1 mg per lg of the cosmetic preparation.
  • the cell aging inhibitor of the present invention can be formulated according to a conventional method.
  • the active ingredient may be simply dissolved or suspended in a pharmaceutically acceptable medium such as physiological buffer saline, but additives commonly used for this may be added.
  • the cell aging of the present invention is simply applied in the cosmetic composition applied to the skin. If the active ingredient of the inhibitor is dissolved or suspended.
  • the electric pulse method and ultrasonic method can be used together.
  • the active ingredient of the cell aging inhibitor is a nucleic acid such as a recombinant vector incorporating antisense RNA, interfering RNA, or antisense cDNA
  • gene transfer is performed to increase the uptake of the nucleic acid into the cell.
  • lipids This makes it easier for the nucleic acid to pass through the lipid bilayer constituting the cell membrane.
  • Various lipids for gene introduction are commercially available. For example, Transfectin (trade name, manufactured by Bio-Rad), Lipofectamine (trade name, manufactured by Invitrogen), and the like can be used.
  • the addition amount of these lipids for gene introduction is not particularly limited, but is usually about 1 ⁇ 1 to 100 ⁇ 1 based on the weight (1 g) of the nucleic acid to be introduced.
  • TIG-7 Normal human fibroblast TIG-7 (available from Human Science Research Resource Bank) was used in this study. The cells were cultured in plastic dishes with 10% urine fetal serum in Dulbecco's modified Eagle medium at 37 ° C, 5% carbon dioxide, and 95% humidity. In this experiment, TIG-7 cells with a population doubling level (PDL) of 32 were used as young cells. TIG-7 cells stop dividing near PDL72 and age.
  • PDL population doubling level
  • GNG-11 cDNA (Genbank NM_004126) is a primer pair (5'-tggacccagtctcaaacttaac-3 (Meki 3 ⁇ 4 No. 3), 5 -cccaagacaaaactttatttgaa-3, Cl cl 3 ⁇ 4 ⁇ No. 4) from total RNA of TIG-7 cells ).
  • cDNA (812 bp) was incorporated into a pGEM-T easy vector (commercially available from Promega), and after sequencing, it was cloned into pCDNA 3.1 (-) (commercially available from Invitrogen) in the sense and antisense orientation.
  • the plasmid (10 ⁇ g) was introduced into the cells using Transfectin (30 ⁇ L) (trade name, commercially available from Bio-Rad). Since the vector contains a neomycin resistance gene, stable transfectants were selected on medium containing G ⁇ SO / zg / mlKA.G. Scientific, Inc.). [0023] 3) Northern blot analysis
  • RNA samples were prepared using an extraction kit. Samples (15 ⁇ g per lane) were electrophoresed on a 1% formaldehyde agarose gel and transferred to a nylon membrane (Hybond-N, Amersham). Membranes were subjected to hybridization with 32p-labeled hnRNPhnRNP (heterogeneous nuclear ribonucleoprotein) Cl / C2 or GAPDH cDNA. Then, it was washed twice with a 0.1XSSC solution containing 0.1% SDS and a 2X SSC solution containing 0.1% SDS (65 ° C., 30 minutes) to prepare autoradiography (FUJI X-ray film).
  • hnRNPhnRNP heterogeneous nuclear ribonucleoprotein
  • BrdU labeling and detection kit II (RocheDiagnostics) were used for the DNA synthesis ability test. Cells were cultured on coverslips to 50% saturation density. Cells were labeled with BrdU at 37 ° C for 30 minutes and fixed with ethyl alcohol at -20 ° C for 30 minutes. The cells were reacted with BrdU antibody for 30 minutes at 37 ° C, then incubated with Alexa Fluor 546 for 2 hours at 37 ° C and observed with a fluorescence microscope.
  • the cells were fixed with 2% formaldehyde / 0.2% glutaraldehyde and stained solution [lm g / ml 5-bromo-4-chloro-3-indolyl-13-D-galactoside, 40 mM citrate phosphate Sodium (pH 6.0), 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 150 mM sodium chloride and 2 mM magnesium chloride] were added and incubated at 35 ° C.
  • Sow cells in a dish were formed. Colonies containing 50 cells or more were counted after staining with methylene blue.
  • GNG11 gene expression was significantly induced in aged TIG-7 cells (Fig. 1) and significantly in resting cells (Fig. 1). Remarkably induced in HeLa cells treated with BrdU. This fact suggests that GNG11 is involved in signal transmission of cellular senescence. Responsiveness to oxidative stress, the main cause of cell aging, was examined (Fig. 2). Young, TIG_7 cells (PDL3
  • a plasmid expressing GNG11 was introduced into young TIG-7 cells (PDL32), and the effect of GNG11 overexpression on cellular senescence was analyzed.
  • Clone G41 8 resistant cells stably incorporating the gene showed cell enlargement and flattening at the colony formation stage and stopped dividing.
  • a plasmid expressing antisense RNA of GNG11 was introduced into TIG-7 cells (62 PDL) immediately before senescence, and the influence on cell senescence was examined. First, colony formation was observed. When the GNG11 gene expression was suppressed, the cells formed normal colonies (Fig. 4). In addition, in cells expressing antisense RNA, disappearance of aging markers and increased DNA synthesis ability (measured using a BrdU labeling kit) were observed. On the other hand, after introducing the plasmid into young TIG-7 cells (32 PDL), G418 resistant clones were selected and their mitotic life was followed (Fig. 5). As a result, anti-RNA-expressing cells showed about 20 prolongation of mitotic life. From the above, it was shown that if the expression of GN G11 is decreased, cell senescence is suppressed and life span is extended.
  • GNG11 most rapidly induced cellular senescence, and antisense RNA inhibited cellular senescence. Induction of cellular senescence can be seen in the activity of Ras and MAP kinase (ERK, p38, JNK is a major component).
  • Ras and MAP kinase ERK, p38, JNK is a major component
  • an effector molecule located downstream of GNG11 may be Ras or a MAP kinase activity factor located upstream of it. High nature. Oxidative stress is thought to activate MAP kinase and induce cellular senescence. Recently, it has been reported that when a membrane receptor works, it generates active oxygen, which activates G-protein.
  • GNG11 responds to active oxygen, it is shown that GNG11 is the most important regulator of cell senescence, and the molecular basis from oxidative stress to cell senescence has been elucidated. GNG11 force It has become possible to control cellular senescence by using derived DNA, RNA, peptides, etc. and natural and artificial compounds that target GNG11. This will lead to the commercialization of pharmaceuticals, cosmetics and health supplements for the purpose of beauty health.
  • the N-terminal region of the GNG11 subunit is also unique in the interaction between subunits that lacks commonality with other components, suggesting a new function of the G protein.
  • GNG11 and GNG1 have high base homology, but the former is expressed in all tissues except the brain, while the latter is suggested to be involved in light transmission in the retina.
  • G protein is dissociated by various stimuli, and GT P-bound ⁇ subunit controls the activity of downstream effector molecules. Recently, it has become clear that a robust ⁇ y complex controls downstream effector molecules (adenyl cyclase subunits II and IV, phospholipase ⁇ 2, phospholipase C subtype, Ca 2+ channel, etc.).

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Abstract

La présente invention concerne un nouvel inhibiteur de vieillissement cellulaire permettant d'inhiber le vieillissement cellulaire de manière efficace et fondamentale. Ledit inhibiteur de vieillissement cellulaire contient, en tant qu'ingrédient actif, une substance permettant d'inhiber l'expression d'un gène codant une sous-unité γ d'une protéine G (11) ou une substance permettant d'inhiber l'effet de vieillissement cellulaire d'une sous-unité γ d'une protéine G (γ sous-unité 11). Étant en mesure de prévenir l'induction du vieillissement cellulaire par un stress par oxydation, cet inhibiteur de vieillissement cellulaire a des applications diverses sur la base du rajeunissement cellulaire. Ledit inhibiteur peut être utilisé en particulier pour développer des produits de beauté ayant des effets anti-vieillissement, des aliments bio, des médicaments, etc.
PCT/JP2007/054513 2006-03-09 2007-03-08 Inhibiteur de vieillissement cellulaire WO2007102572A1 (fr)

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JP2008503904A JPWO2007102572A1 (ja) 2006-03-09 2007-03-08 細胞老化抑制剤

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JP2006-064955 2006-03-09
JP2006064955 2006-03-09

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WO2007102572A1 true WO2007102572A1 (fr) 2007-09-13

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JP (1) JPWO2007102572A1 (fr)
WO (1) WO2007102572A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011045334A (ja) * 2009-08-28 2011-03-10 Kyushu Univ 細胞老化関連因子
KR20180080494A (ko) * 2017-01-04 2018-07-12 연세대학교 산학협력단 세포 노화 억제용 또는 피부 노화 개선용 조성물
US10226441B2 (en) 2014-12-09 2019-03-12 Nihon Sizen Hakkoh Co., Ltd. Aging inhibitor

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113699182B (zh) * 2021-08-31 2023-09-15 西南大学 沉默桑实杯盘菌G蛋白γ亚基编码基因CsGγ的沉默载体及其应用和方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BALCUEVA E.A. ET AL.: "Human G Protein gamma11 and gamma14 Subtypes Define a New Functional Subclass", EXPERIMENTAL CELL RESEARCH, vol. 257, no. 2, 2000, pages 310 - 319, XP003017625 *
DHANASEKARAN N. ET AL.: "G protein subunits and cell proliferation", BIOLOGICAL, SIGNALS AND RECEPTORS, vol. 7, no. 2, 1998, pages 109 - 117, XP003017626 *
HOSSAIN M.N. ET AL.: "G-protein gamma subunit GNG11 strongly regulates cellular senescence", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 351, no. 3, 22 December 2006 (2006-12-22), pages 645 - 650, XP005726083 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011045334A (ja) * 2009-08-28 2011-03-10 Kyushu Univ 細胞老化関連因子
US10226441B2 (en) 2014-12-09 2019-03-12 Nihon Sizen Hakkoh Co., Ltd. Aging inhibitor
KR20180080494A (ko) * 2017-01-04 2018-07-12 연세대학교 산학협력단 세포 노화 억제용 또는 피부 노화 개선용 조성물

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