WO2018130178A1 - Small molecule compound/combination for preventing, delaying, or reversing aging of cell, tissue, organ, or body, product and use thereof - Google Patents

Small molecule compound/combination for preventing, delaying, or reversing aging of cell, tissue, organ, or body, product and use thereof Download PDF

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WO2018130178A1
WO2018130178A1 PCT/CN2018/072228 CN2018072228W WO2018130178A1 WO 2018130178 A1 WO2018130178 A1 WO 2018130178A1 CN 2018072228 W CN2018072228 W CN 2018072228W WO 2018130178 A1 WO2018130178 A1 WO 2018130178A1
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inhibitor
small molecule
cells
combination
agonist
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胡敏
李燕皎
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昆明学院
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Definitions

  • the present invention relates to cell biology, tissue engineering and medicine, and more particularly to a small molecule compound/combination, product and use thereof for preventing, delaying or reversing cell, tissue, organ, body aging.
  • the present invention provides a small molecule compound/combination, product and use thereof for preventing, delaying or reversing cell, tissue, organ, and body aging.
  • the small molecule compound combination has regulation, integrity, reversibility and safety in the process of anti-aging of cells, tissues, organs and organisms.
  • a combination of small molecule compounds for preventing, delaying or reversing the aging process of cells, tissues, organs or organisms of a mammal such as a human, the combination of small molecule compounds comprising a DNMT inhibitor, an HMT inhibitor, a group At least one of a protein demethylase inhibitor, a TGF- ⁇ inhibitor, a WNT/ ⁇ -catenin agonist, a cAMP agonist, and a lysine deacetylase inhibitor.
  • the small molecule compound is combined into at least one of a DNMT inhibitor, an HMT inhibitor, a histone demethylase inhibitor, and a lysine deacetylase inhibitor.
  • the small molecule compound is combined into at least one of a TGF- ⁇ inhibitor, a WNT/ ⁇ -catenin agonist, a cAMP agonist, a DNMT inhibitor, an HMT inhibitor, and a lysine deacetylase inhibitor. .
  • the small molecule compound further includes at least one of an RAR agonist, an ascorbate (ascorbic acid), and a ROCK inhibitor.
  • the small molecule compound combination comprises a first stage compound and a second stage compound which are used in stages according to a time series, the first stage compound being a DNMT inhibitor, an HMT inhibitor, a histone demethylase inhibitor At least one of a T GF- ⁇ inhibitor, a WNT/ ⁇ -catenin agonist, a cAMP agonist, and a lysine deacetylase inhibitor;
  • the second stage compound is a DNMT inhibitor, an HMT inhibitor, a histone demethylase inhibitor, a TGF- ⁇ inhibitor, a WNT/ ⁇ -catenin agonist, a cAMP agonist, a lysine deacetylase At least one of an inhibitor, a RAR agonist, an ascorbate (ascorbic acid), and a ROCK inhibitor.
  • the TGF-beta receptor inhibitor is a type I TGF-beta receptor inhibitor and the cAMP agonist is an EPAC/RAP1 agonist.
  • the small molecule compound combination is used in combination with PDGF-AB to improve the efficiency of the action of the small molecule compound combination.
  • the lysine deacetylase inhibitor comprises sodium phenylbutyrate, butyrate, sodiu m butyrate, MC1568, CI994 (Tacedinaline), chidamide, CAY10683 (SantacruzaMate A), CUD C-907, M344 (Histone Deacetylase Inhibitor III) ), LAQ824 (NVP-LAQ824, Dacinostat), Prac inostat (SB939), VPA (Valproic acid), Valproic acid sodium salt, Scriptaid, Apicidin, LBH-589 (Panobinostat), MS-275, SAHA (Vorinostat), Trichostatin ( TSA), Psammaplin A, PCI-24781 (A bexinostat), Rocilinostat (ACY-1215), Mocetinostat (MGCD0103), 4-Phenylbutyrate (4PB), sp litomicin, SRT1720, resveratrol
  • the TGF-beta receptor inhibitors include 616452, LY2109761, Pirfenidone, Repox (E-616452), SB431542, A77-01, Tranilast, Galunisertib (LY2157299), A8301, GW788388, ITD-1, SD208, S B525334, LY364947 At least one of ASP3029, D4476 and SB505124;
  • the WNT/ ⁇ -catenin agonists include MAY-262611, CHIR98014, CHIR99021, LiCl, Li 2 CO 3 , TD114-2, AZD2858, AZD1080, BIO, Kenpaullone, TWS119, LY2090314, CBM1078, SB216763 and AR-A014418 At least one
  • the cAMP agonist comprises at least one of an EPAC/RAP1 agonist, 8-Bromo-cAMP, Dibutyryl-Camp and Sp-8-Br-cAMPs;
  • the EPAC/RAP1 agonists include Forskolin, IBMX, Prostaglandin E2 (PGE2), NKH477, 8-pCPT-2'-O-Me-cAMP, GSK256066, Apremilast (CC-10004), Roflumilast, Cilomilast, Rolip ram and Milrinone At least one of them;
  • the RAR agonist comprises at least one of TTNPB, Bexarotene, Ch55, Tamibarotene, Retinol, AM580, AT RA, 13-cis RA, Vitamin A and Vitamin A derivatives;
  • the ROCK inhibitor comprises at least one of Y-27632, Y-27632 2HCl, Thiazovivin, Ripasudil (K-115), Fasu dil, Fasudil (HA-1077) HCl, GSK429286A, RKI-1447 and PKI-1313;
  • the DNMT inhibitor comprises at least one of RG108, Thioguanine, 5-Aza-2'-deoxycytidine (Decitabine), SGI-1027, Zebularine and 5-Azacytidine (AZA);
  • the HMT inhibitor comprises at least one of EPZ004777, EPZ5676, GSK503, BIX 01294, DZNep, DZNep HCL, SGC 0946;
  • the histone demethylase inhibitor comprises at least one of parnate (tranylcypromine), Tranylcypromine (2-PCPA) HCl SP2509, 4SC-202, ORY-1001 (RG-6016), GSKJ1 and GSK-LSD1.
  • the lysine deacetylase inhibitor is at least one of sodium butyrate, VPA and Trichostatin (T SA);
  • the TGF- ⁇ receptor inhibitor is at least one of Repx (E-616452) and SB431542;
  • the WNT/ ⁇ -catenin agonist is at least one of CHIR99021 and BIO;
  • the cAMP agonist is at least one of Forskolin and Rolipram;
  • the RAR agonist is at least one of TTNPB and AM580;
  • the ROCK inhibitor comprises at least one of Y-27632 and Thiazovivin;
  • the DNMT inhibitor is at least one of RG108, 5-Aza-2'-deoxycytidine (Decitabine) and 5-Azacytidine (AZA);
  • the HMT inhibitor is at least one of EPZ004777, EPZ5676 and SGC 0946;
  • the inhibitor of the histone demethylase is at least one of parnate (tranylcypromine) and Tranylcypromine (2-PCPA) HCl.
  • the combination of small molecule compounds is used to prolong the telomere length of mammalian cells such as humans, transiently stimulate the expression or up-regulation of telomere-associated proteins, such as TERT (telomerase reverse transcriptase) expression and TPP1 up-regulation; It does not stimulate mammalian cells such as humans to express dry genes such as OCT4, Nanog, and the like.
  • TERT telomerase reverse transcriptase
  • the effective concentration of the specific small molecule compound is as follows, the concentration range given below is only a reference, and can be adapted on this basis, if other small molecules replace the following small molecules, the concentration can also be adapted. Adjustment.
  • Forskolin concentration is 2 ⁇ M ⁇ 20 ⁇ M; Repsox concentration is 2 ⁇ 15uM; CHIR99021 concentration is 1 ⁇ M ⁇ 10 ⁇ M; VPA concentration is 0.5mM ⁇ 1.5mM; TTNPB concentration is 3 ⁇ M ⁇ 8 ⁇ M; AM580 concentration is 0.03 ⁇ 0.08 ⁇ M; EPZ004777 concentration is 3 ⁇ 8 ⁇ M; Y-27632 concentration is 3-15 ⁇ M, L-Ascorbin acid 2-phosphate concentration is 0.15-0.25 mM; 5-Aza-2'-deoxycytidine concentration is 0.5-20 ⁇ M.
  • the mechanism of the present invention for preventing, delaying or reversing aging is as follows: the present invention by epigenetical regulation of aging or senescent cells, or by activating early development-associated transcription factors, such as activation of the WNT/ ⁇ -catenin signaling pathway, Activation of cAMP signaling pathway, agonistic RA signaling pathway, inhibition of TGF-beta signaling pathway, prevention, delay or reversal of cellular senescence.
  • telomere length in mammalian cells such as humans, transiently stimulate expression/upregulation of telomere-associated proteins, such as TERT (telomerase reverse transcriptase) and TPP1, without stimulating mammals
  • telomere-associated proteins such as TERT (telomerase reverse transcriptase) and TPP1
  • human cells express dry genes, such as OCT4, Nanog, etc., safe and effective.
  • kits for treating small molecule compounds.
  • a use of the small molecule compound combination for preventing, delaying or reversing a cell, tissue, organ or body aging process and related diseases of a mammal such as a human, and for improving a mammal such as a human tissue or an organ The application of repair capabilities.
  • the cells are derived from a mammalian cell such as a human body or a cell cultured in vitro.
  • a method for preventing, delaying or reversing the aging process of a cell, tissue, organ or body of a mammal such as a human by the combination of the small molecule compound is provided.
  • a younger cell and a cell product obtained by a combination of the small molecule compounds are provided.
  • aging as used in this specification has the same meaning as “aging”, and younger cells refer to cells before aging. However, the “aging” described in the present invention does not include cells that permanently stop dividing.
  • the invention has the following beneficial effects: the invention adopts a small molecule compound or a combination thereof to prevent, delay or reverse the aging of cells, tissues, organs and organisms, prolong the telomere length of the cells, and has a clear target, clear composition, stable and stable, and can be accurately regulated. Anti-aging process; and small molecule compounds are easy to control and scale production. Because the body or cells do not express dry genes during the process, the safety is high; and the small molecule compounds and their combinations are easy to absorb, which can prevent, delay and reverse the cells, tissues, organs and organisms. Aging is of great significance in delaying the process of human aging, improving health and quality of life.
  • Figure 1 is a view showing the morphology of human skin fibroblasts after the action of small molecule compounds
  • FIG. 2 is a morphology diagram of human bone marrow mesenchymal stem cells after treatment with a small molecule compound
  • Figure 3 is a diagram showing the identification of human dermal fibroblast cell traits after the action of small molecule compounds
  • Figure 4 shows the identification of cell traits of human bone marrow mesenchymal stem cells after treatment with small molecule compounds
  • Figure 5 is a graph showing changes in telomere length of human cells after treatment with small molecule compounds
  • Figure 6 shows the expression of telomere-associated genes in human cells after treatment with small molecule compounds
  • Figure 7 is a graph showing changes in expression of aging-related genes in human dermal fibroblasts after treatment with small molecule compounds
  • Figure 8 is a graph showing the results of in vitro functional viability of human skin fibroblasts after action of small molecule compounds
  • Figure 9 is a graph showing the results of in vivo functional activity of human skin fibroblasts after action of small molecule compounds.
  • Figure 10 is a graph showing the results of detection and tumorigenicity of cell dryness after long-term passage of a small molecule compound
  • Figure 11 is a clonal formation and morphological map of human dermal fibroblasts after long-term passage of small molecule compounds.
  • a skin tissue block of about 1 cm in diameter from a volunteer separating the skin fibroblasts by adherence method, and separating the cells into a basic culture solution: 10% fetal bovine serum (Hyclone) + 100 U/ Ml penicillin (Sigm a) + 100 ⁇ g / ml streptomycin (Sigma) + high sugar DMDM.
  • the cell is the 12th generation, the day before treatment (Day-1), the inoculated cell density is 1 ⁇ 2.5 ⁇ 10 4 /cm 2 and cultured at 37 ° C, 5% CO 2 in the incubator.
  • the basal medium as a small molecule treatment solution, culture the cells for 2-20 days, and the small molecule treatment solution means: 10% fetal bovine serum (H yclone) + 100 U / ml penicillin (Sigma) + 100 ⁇ g / ml streptomycin (Sigma + high glucose DMDM medium (Gibco) + f orskolin (2 ⁇ M ⁇ 25 ⁇ M) + Repx (2 ⁇ 15uM) + CHIR99021 (1 ⁇ M ⁇ 10 ⁇ M) + VPA (0.5mM ⁇ 1.5mM) + 5-Aza-2'-deoxycytidine (0.5-20 ⁇ M), 10% fetal bovine serum in this culture system can also be replaced by serum replacement (invitrogen) at a concentration of 10% to 20%; 100 U/ml penicillin (Sigma) and 100 ⁇ g/ml streptomycin (Sig) Ma) can not be used.
  • the cells were cultured at 37 ° C in a 5% CO 2 atmosphere.
  • the basal medium is: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 ⁇ g / ml streptomycin (Sigma) + high sugar DMDM, 10% fetal bovine serum can also be replaced by serum ( Invitrogen) is replaced by a concentration of 10% to 20%; 100 U/ml penicillin (Sigma) and 100 ⁇ g/ml streptomycin (Sigma) may not be used.
  • the cells were passaged to the 14th generation, and stained with e. coli CD29-PE flow antibody.
  • the control was the same type of reference substance of the same manufacturer, and the analysis was performed on the BD JAZZ flow meter according to the manufacturer's instructions.
  • the cells were passaged to the 14th passage, cultured in a 24-well plate, and the cells were fixed with 4% PFA for 10 minutes, and stained with the Vimentin antibody of Santa cruz according to the procedure of immunofluorescence;
  • the amount of cells was inoculated with 1.5 ⁇ 10 5 as the initial, the cells were counted for 12 consecutive days, and 3 groups were repeated at each time point;
  • telomere length was detected by the Cawthon qPCR method.
  • the fluorescent dye was Takara SYBR Green I.
  • mTel CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT;
  • 36B4-S-F CAGCAAGTGGGAAGGTGTAATCC
  • TERT was detected by PCR.
  • the primer sequence of human TERT was F: 5'-CGGAAGAGTGTCTGGAGCAA-3; R5'-GGATGAAGCGGAGTCTGGA-3';
  • RNA samples were collected, and the expression levels of genes TPP1, CDKN1A, ATF3, GADD45B, BTG2, H2AFX, ITGA6, COL1A1 were quantified by transcriptome analysis;
  • the experimental group is a small molecule compound treatment cell group
  • the untreated cell group the PBS group, which is passaged to the 14th generation, and the animal experiment is performed according to the requirements of the animal ethics committee
  • the donor C57B/6 mouse is anesthetized, supine Fixed, full-back hair removal, 1.5cm x 1.5cm full-thickness skin on the back, remove subcutaneous fat and blood vessels, cut the skin of the mouse into a 1.0cm-diameter skin, and place it in a dish containing sterile PBS. .
  • the cut piece of skin was turned over and placed in a petri dish with physiological saline, and the subcutaneous tissue was gently cut into the dermis with a surgical blade, and then placed in sterile physiological saline for use.
  • the recipient was BALB/c mice, the back was depilated after anesthesia, the iodine disinfected the back skin, a skin gap of 1.0 cm in diameter was prepared, the blood vessels were preserved, and the C57B/6 skin was transplanted in the reverse hair direction, and the surface was covered with Vaseline oil yarn. And band-aid, moderately tight.
  • Each group of liquids in the list was injected from the tail vein within 1 hour after transplantation.
  • the cells are cultured in a basic culture medium: 8% fetal bovine serum (Hyclone) + 100 U/ml penicillin (Sigma) + 100 ⁇ g/ml streptomycin (Sigma) + low sugar DMDM.
  • a basic culture medium 8% fetal bovine serum (Hyclone) + 100 U/ml penicillin (Sigma) + 100 ⁇ g/ml streptomycin (Sigma) + low sugar DMDM.
  • the cell is the 10th generation, the day before treatment (Day-1), the inoculated cell density is 1 ⁇ 2.5 ⁇ 10 4 /cm 2 and cultured at 37 ° C, 5% CO 2 in the incubator.
  • the basal medium as a small molecule treatment solution, culture the cells for 2-20 days, and the small molecule treatment solution means: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 ⁇ g / ml streptomycin (Sigma) + high glucose DMDM medium (Gibco) + forskolin (2 ⁇ M ⁇ 25 ⁇ M) + Repsox (2 ⁇ 15uM) + CHIR99021 (1 ⁇ M ⁇ 10 ⁇ M) + VPA (0.5mM ⁇ 1.5mM), 10% fetal bovine serum in the culture system It can be replaced by a serum substitute (invitrogen) at a concentration of 10% to 20%; 100 U/ml penicillin (Sigma) and 100 ⁇ g/ml streptomycin (Sigma) may not be used.
  • the cells were cultured at 37 ° C in a 5% CO 2 atmosphere.
  • the basal culture solution was completely replaced, the culture was maintained normally, and the cells were cultured at 37 ° C in a 5% CO 2 atmosphere.
  • the basal medium 8% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 ⁇ g / ml streptomycin (Sigma) + low sugar DMDM, 10% fetal bovine serum can also be replaced by serum (invitrogen ) is replaced by a concentration of 10% to 20%; 100 U/ml penicillin (Sigma) and 100 ⁇ g/ml streptomycin (Sigma) may not be used.
  • the amount of cells was inoculated with 1.5 ⁇ 10 5 as the initial, the cells were counted for 12 consecutive days, and 3 groups were repeated at each time point;
  • telomere length was detected by the Cawthon qPCR method.
  • the fluorescent dye was Takara SYBR Green I.
  • mTel CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT;
  • 36B4-S-F CAGCAAGTGGGAAGGTGTAATCC
  • TERT was detected by PCR.
  • the primer sequence of human TERT was F: 5'-CGGAAGAGTGTCTGGAGCAA-3; R5'-GGATGAAGCGGAGTCTGGA-3';
  • 4.7 cells were passaged to the 14th generation, RNA samples were collected, and the expression levels of genes TPP1, CDKN1A, ATF3, GADD45B, BTG2, and H2AFX were quantified by transcriptome analysis;
  • the experimental group is a small molecule compound treatment cell group
  • the untreated cell group the PBS group, which is passaged to the 14th generation, and the animal experiment is performed according to the requirements of the animal ethics committee
  • the donor C57B/6 mouse is anesthetized, supine Fixed, full-back hair removal, 1.5cm x 1.5cm full-thickness skin on the back, remove subcutaneous fat and blood vessels, cut the skin of the mouse into a 1.0cm-diameter skin, and place it in a dish containing sterile PBS. .
  • the cut piece of skin was turned over and placed in a petri dish with physiological saline, and the subcutaneous tissue was gently cut into the dermis with a surgical blade, and then placed in sterile physiological saline for use.
  • the recipient was BALB/c mice, the back was depilated after anesthesia, the iodine disinfected the back skin, a skin gap of 1.0 cm in diameter was prepared, the blood vessels were preserved, and the C57B/6 skin was transplanted in the reverse hair direction, and the surface was covered with Vaseline oil yarn. And band-aid, moderately tight.
  • Each group of liquids in the list was injected from the tail vein within 1 hour after transplantation.
  • a skin tissue block of about 1 cm in diameter from a volunteer separating the skin fibroblasts by adherence method, and separating the cells into a basic culture solution: 10% fetal bovine serum (Hyclone) + 100 U/ Ml penicillin (Sigm a) + 100 ⁇ g / ml streptomycin (Sigma) + high sugar DMDM.
  • the cell is the 12th generation, the day before treatment (Day-1), the inoculated cell density is 1 ⁇ 2.5 ⁇ 10 4 /cm 2 and cultured at 37 ° C, 5% CO 2 in the incubator.
  • the basal medium as a small molecule treatment solution, culture the cells for 2-20 days, and the small molecule treatment solution means: 10% fetal bovine serum (H yclone) + 100 U / ml penicillin (Sigma) + 100 ⁇ g / ml streptomycin (Sigma + high glucose DMDM medium (Gibco) + f orskolin (2 ⁇ M ⁇ 25 ⁇ M) + 5-Aza-2'-deoxycytidine (0.5 ⁇ 20 ⁇ M), 10% fetal bovine serum in this culture system can also be replaced by serum (invitrogen ) is replaced by a concentration of 10% to 20%; 100 U/ml penicillin (Sigma) and 100 ⁇ g/ml streptomycin (Sigma) may not be used.
  • the cells were cultured at 37 ° C in a 5% CO 2 atmosphere.
  • the basal medium is: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 ⁇ g / ml streptomycin (Sigma) + high sugar DMDM, 10% fetal bovine serum can also be replaced by serum ( Invitrogen) is replaced by a concentration of 10% to 20%; 100 U/ml penicillin (Sigma) and 100 ⁇ g/ml streptomycin (Sigma) may not be used.
  • a skin tissue block of about 1 cm in diameter from a volunteer separating the skin fibroblasts by adherence method, and separating the cells into a basic culture solution: 10% fetal bovine serum (Hyclone) + 100 U/ Ml penicillin (Sigm a) + 100 ⁇ g / ml streptomycin (Sigma) + high sugar DMDM.
  • the cell is the 12th generation, the day before treatment (Day-1), the inoculated cell density is 1 ⁇ 2.5 ⁇ 10 4 /cm 2 and cultured at 37 ° C, 5% CO 2 in the incubator.
  • the small molecule treatment solution means: 10% fetal bovine serum (H yclone) + 100 U / ml penicillin (Sigma) + 100 ⁇ g / ml streptomycin (Sigma + high glucose DMDM medium (Gibco) + V PA (0.5 mM ⁇ 1.5 mM) + Repx (2 ⁇ 15 uM), 10% fetal bovine serum in this culture system can also be 10% ⁇ by serum substitute (invitrogen) 20% concentration substitution; 100 U/ml penicillin (Sigma) and 100 ⁇ g/ml streptomycin (Sig ma) may not be used.
  • the cells were cultured at 37 ° C in a 5% CO 2 atmosphere.
  • the basal medium is: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 ⁇ g / ml streptomycin (Sigma) + high sugar DMDM, 10% fetal bovine serum can also be replaced by serum ( Invitrogen) is replaced by a concentration of 10% to 20%; 100 U/ml penicillin (Sigma) and 100 ⁇ g/ml streptomycin (Sigma) may not be used.
  • the small molecule compound of the skin fibroblasts is treated as follows: the base culture solution is replaced with a small molecule treatment solution, and the cells are cultured for 2-20 days, and the small molecule treatment solution means: 10% fetal bovine serum (Hyclone) + 100 U/ Ml penicillin (Sigma) + 100 ⁇ g / ml streptomycin (Sigma) + high glucose DMDM medium (Gibco) + VPA (0.5 mM ⁇ 1.5 mM) + Repx (2 ⁇ 15u M) + 5-Aza-2'-deoxycytidine (0.5-20 ⁇ M), 10% fetal bovine serum in this culture system can also be replaced by serum substitute (in vitrogen) at a concentration of 10% to 20%; 100 U/ml penicillin (Sigma) and 100 ⁇ g/ml streptomycin ( Sigma) can not be used. Cells were cultured at 37 °C, 5% CO 2 environment. The remaining steps are the same as in the first embodiment.
  • the small molecule compound of the skin fibroblasts is treated as follows: the base culture solution is replaced with a small molecule treatment solution, and the cells are cultured for 2-20 days, and the small molecule treatment solution means: 10% fetal bovine serum (Hyclone) + 100 U/ Ml penicillin (Sigma) + 100 ⁇ g / ml streptomycin (Sigma) + high glucose DMDM medium (Gibco) + VPA (0.5 mM ⁇ 3 mM), 10% fetal bovine serum in this culture system can also be replaced by serum (invitrogen ) is replaced by a concentration of 10% to 20%; 100 U/ml penicillin (Sigm a) and 100 ⁇ g/ml streptomycin (Sigma) may not be used.
  • the cells were cultured at 37 ° C in a 5% CO 2 atmosphere. The remaining steps are the same as in the first embodiment.
  • the small molecule compound of the skin fibroblasts is treated as follows: the base culture solution is replaced with a small molecule treatment solution, and the cells are cultured for 2-20 days, and the small molecule treatment solution means: 10% fetal bovine serum (Hyclone) + 100 U/ Ml penicillin (Sigma) + 100 ⁇ g / ml streptomycin (Sigma) + high glucose DMDM medium (Gibco) + 5-Aza-2'-deoxycytidine (0.2 ⁇ 20 ⁇ M), 10% fetal bovine serum can also be used in the culture system It is replaced by a serum substitute (invitrogen) at a concentration of 10% to 20%; 100 U/ml penicillin (Sigma) and 100 ⁇ g/ml streptomycin (Sigma) may not be used.
  • the cells were cultured at 37 ° C in a 5% CO 2 atmosphere. The remaining steps are the same as in the first embodiment.
  • the small molecule compound of the skin fibroblasts is treated as follows: the base culture solution is replaced with a small molecule treatment solution, and the cells are cultured for 2-20 days, and the small molecule treatment solution means: 10% fetal bovine serum (Hyclone) + 100 U/ Ml penicillin (Sigma) + 100 ⁇ g / ml streptomycin (Sigma) + high sugar DMDM medium (Gibco) + RG108 (0.5 ⁇ 30 ⁇ M), 10% fetal bovine serum in this culture system can also be replaced by serum (invitrogen) Replaced at a concentration of 10% to 20%; 100 U/ml penicillin (Sigma) and 100 ⁇ g/ml streptomycin (Sigma) may not be used.
  • the cells were cultured at 37 ° C in a 5% CO 2 atmosphere. The remaining steps are the same as in the first embodiment.
  • the small molecule compound of the skin fibroblasts is treated as follows: the base culture solution is replaced with a small molecule treatment solution, and the cells are cultured for 2-20 days, and the small molecule treatment solution means: 10% fetal bovine serum (Hyclone) + 100 U/ Ml penicillin (Sigma) + 100 ⁇ g / ml streptomycin (Sigma) + high glucose DMDM medium (Gibco) + Repsox (2 ⁇ 15 uM) + CHIR99021 (1 ⁇ M ⁇ 10 ⁇ M) + VPA (0.5 mM ⁇ 1.5 mM) + TTNPB ( 3 ⁇ M ⁇ 8 ⁇ M)+AM580(0.03 ⁇ 0.08 ⁇ M)+EPZ004777(3 ⁇ 8 ⁇ M)+Y-27632(3 ⁇ 15 ⁇ M)+L-Ascorbin acid 2-phosphate(0.15 ⁇ 0.25m M), 10% in the culture system Fetal bovine serum can also be replaced by a serum replacement (invitrogen) at
  • the small molecule compound of the skin fibroblasts is treated as follows: the base culture solution is replaced with a small molecule treatment solution, and the cells are cultured for 2-20 days, and the small molecule treatment solution means: 10% fetal bovine serum (Hyclone) + 100 U/ Ml penicillin (Sigma) + 100 ⁇ g / ml streptomycin (Sigma) + high sugar DMDM medium (Gibco) + forskolin (2 ⁇ M ⁇ 25 ⁇ M) + Repso x (2 ⁇ 15 uM) + CHIR99021 (1 ⁇ M ⁇ 10 ⁇ M) + VPA (0.5 mM ⁇ 1.5 mM) + PDGF-AB (100 ⁇ 250 ng / mL), 10% fetal bovine serum in this culture system can also be replaced by serum replacement (invitrogen) at a concentration of 10% to 20%; 100 U / ml penicillin ( Sigma) and 100 ⁇ g/ml streptomycin (Sigma)
  • the small molecule compound of the bone marrow mesenchymal stem cells is treated as follows: the base medium is replaced with a small molecule treatment solution, and the cells are cultured for 2-20 days, and the small molecule treatment solution means: 10% fetal bovine serum (Hyclone) + 100U.
  • the culture system The 10% fetal bovine serum can also be replaced by a serum replacement (invitrogen) at a concentration of 10% to 20%; 100 U/ml penicillin (Sigma) and 100 ⁇ g/ml streptomycin (Sigma) may not be used.
  • Cells were cultured at 37 °C, 5% CO 2 environment.
  • Fig. 1 shows the morphological changes of human skin fibroblasts after the action of small molecule compounds, and when the A picture is not treated, the normal cultured skin fibroblasts are passed to the 13th.
  • Generation some fibroblasts that originally grew in long spindle shape began to appear with large cell size and irregular cell shape, stained with ⁇ -galactosidase, and B showed senescent cells stained blue;
  • the cells of the 14th generation were substantially spindle-shaped in a regular shape, and stained with ⁇ -galactosidase, and the result was negative.
  • Figure 2 shows the changes in the morphology of human bone marrow mesenchymal stem cells after the action of small molecule compounds.
  • the normal cultured mesenchymal stem cells are passed to the 10th generation, and some cell bodies with original spindle-shaped growth become larger. , cell elongated or irregular growth, stained with ⁇ -galactosidase, B picture shows senescent cells stained blue; after treatment with the method as in Example 2, the 12th generation of cells, the basic rules The ground was spindle-shaped and stained with ⁇ -galactosidase, and the result was negative.
  • FIG 3 is a diagram showing the cell traits of human dermal fibroblasts after the action of small molecule compounds. After treatment with the method of Example 1, the cells were normally expressed as the molecular marker CD29 of skin fibroblasts (Fig. A). Vimentin (panel B); Figure C compares the value-added of cells before and after treatment. The value-added ability of Fib-treated in the treatment group is more obvious than that of untreated Fib.
  • Figure 4 is a diagram showing the cell traits of human bone marrow mesenchymal stem cells after the action of small molecule compounds.
  • Figure A shows the expression of molecular markers of mesenchymal stem cells before and after treatment, and positive expression of CD29, CD90, CD73. And the negative CD34, CD45 expression in the cells before and after treatment; B-treated cells after treatment MSC-treated better than the pre-treatment MSC value-added; C picture shows the treated MSC cells, also normal 3 series Differentiation, as shown in the figure, shows osteogenesis and adipogenic differentiation of the cells.
  • telomere length is a change in the telomere length of a human-derived cell by a small molecule compound.
  • the human skin fibroblasts before treatment are used as a control, and the methods in Example 1 and Examples 3 to 10 are used.
  • the telomere length was increased by more than 1.5 times; human bone marrow mesenchymal stem cells were treated in panel B, and the length of the back end granules was increased by the methods of Example 2 and Example 11.
  • Figure 6 is a comparison of telomere length and age of human cells after treatment with small molecule compounds, and expression of telomere-associated genes: compared with mean telomere length in healthy populations of about 35 years old, and compared with autologous After treatment with small molecules, the telomere length of the individual-derived skin fibroblasts increased significantly, as shown in Figure A; the transiently high expression of the gene TPP1 associated with telomere elongation, as shown in Figure B; Figure C shows that TERT ( Telomerase reverse transcriptase) in immortalized cells 293T (“1"), Day12 Fib (human dermal fibroblasts; "2", Example 1), Day12 MSC (human bone marrow mesenchymal stem cells) "3", transient expression in Example 2).
  • TERT Telomerase reverse transcriptase
  • Figure 7 shows the expression changes of aging-related genes in human dermal fibroblasts after treatment with small molecule compounds.
  • the genes related to aging are down-regulated, for example, the gene CDKN1A associated with the P53 tumor suppressor pathway, ATF3, GADD45B, BTG2, H2A histone family member X associated with aging;
  • Figure 8 is a graph showing the in vitro functional viability of human dermal fibroblasts after action of a small molecule compound: as in Example 3, type I collagen of skin fibroblasts was significantly up-regulated after treatment; ITGA6, a marker for early developmental skin cells, The up-regulation is obvious after processing.
  • Figure 9 is a test for the functional viability of human dermal fibroblasts in vivo after the action of small molecule compounds: fibroblasts isolated from the skin have weak immunomodulatory ability, and after treatment of cells by the method of Example 6, transplantation in allogeneic skin
  • the culture solution after 48 hours of cell culture was injected into the recipient mice, and the skin graft site was observed 8 days later, and the donor skin was immunized compared with the control group (untreated cell group Fib, PBS group).
  • the rejection is small, and the treated cells have obvious systemic immunomodulatory effects.
  • Fig. 10 is a graph showing the results of detection and tumorigenicity of cell dryness after long-term passage of a small molecule compound: For the cells treated in Example 1, subcutaneous cell transplantation of Nod-SCID mice was performed, and no effect was observed after 28 days. Tumors are shown in the white circle of Figure A; in Figure B, the cells were stained with the dry gene OCT4 and no expression was observed.
  • Figure 11 After the action of the small molecule compound (as in Example 1), the number of clones of human skin fibroblasts was higher than that before treatment, as shown in Figure A; Figure B shows the cloned form, clones of pre-treatment cells (Fib) Smaller, cell-dispersed; treated cells (Fib-treated) are tightly packed and larger clones.

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Abstract

A small molecule compound/combination for preventing, delaying, or reversing the aging of a cell, a tissue, an organ, or a body, and use thereof. The small molecule compound combination comprises at least one of a DNMT inhibitor, an HMT inhibitor, a histone demethylase inhibitor, a TGF-β inhibitor, a WNT/β-catenin agonist, a cAMP agonist, a lysine deacetylase inhibitor, an RAR agonist, and a ROCK inhibitor.

Description

用于预防、延缓或逆转细胞、组织、器官、机体衰老的小分子化合物/组合，产品及其用途Small molecule compounds/combinations, products and uses thereof for preventing, delaying or reversing cell, tissue, organ, and body aging 技术领域Technical field
本发明涉及细胞生物学，组织工程和医学，尤其涉及一种用于预防、延缓或逆转细胞、组织、器官、机体衰老的小分子化合物/组合，产品及其用途。The present invention relates to cell biology, tissue engineering and medicine, and more particularly to a small molecule compound/combination, product and use thereof for preventing, delaying or reversing cell, tissue, organ, body aging.
背景技术Background technique
人类机体衰老是现代社会的主要健康问题，也是大部分老年性疾病的主要原因。衰老过程伴随身体机能的降低、对外界变化适应能力的减弱、代偿功能的低下，最终诱发多种疾病。如心血管疾病、恶性肿瘤、神经、内脏、骨骼和肌肉的退行性疾病、糖尿病等都与衰老密切相关。年龄每增加10岁，因心血管病的病死率即增加2～3倍，可见衰老对人类生活质量与健康状态影响巨大。Human body aging is a major health problem in modern society and a major cause of most senile diseases. The aging process is accompanied by a decrease in physical function, a weakening of adaptability to external changes, and a low compensatory function, eventually leading to various diseases. Degenerative diseases such as cardiovascular disease, malignant tumors, nerves, internal organs, bones and muscles, and diabetes are all closely related to aging. For every 10 years of age, the mortality rate of cardiovascular disease is increased by 2 to 3 times. It can be seen that aging has a great impact on human quality of life and health.
近几十年来，与衰老相关的因素(如衰老相关基因)以及抗衰老药物、方法不断被报道。例如2016年，美国梅奥诊所的研究团队通过清除衰老细胞，使小鼠的寿命延长超过20％；不同的研究机构发现通过喂食小鼠雷帕霉素、辅酶I、NMN(烟酰胺单核苷酸)、亚精胺等可以显著延长小鼠的寿命；有研究机构利用线虫模型揭示了RNA剪接(RNA splicing)功能与长寿之间的关联；另有研究机构报道了HOXA9在老年时的重激活会限制骨骼肌的再生，NRF2的激活有利于抗衰老；另外，据有关报道，清除线粒体中突变的DNA,或调整跑步、节食等生活习惯与生活方式对延缓衰老也有积极作用。In recent decades, factors related to aging (such as aging-related genes) and anti-aging drugs and methods have been reported. For example, in 2016, the Mayo Clinic research team extended the lifespan of mice by more than 20% by removing senescent cells; different research institutions found that by feeding mice rapamycin, coenzyme I, NMN (nicotinamide mononucleoside) Acids, spermidine and the like can significantly prolong the lifespan of mice; some institutions have used the nematode model to reveal the relationship between RNA splicing function and longevity; another research institute reported the reactivation of HOXA9 in old age. It will limit the regeneration of skeletal muscle. The activation of NRF2 is beneficial to anti-aging. In addition, according to reports, clearing the mutated DNA in mitochondria, or adjusting lifestyle and lifestyle such as running and dieting, also have a positive effect on delaying aging.
尽管上述的物质或者方法都有改善器官健康状态，从而提高寿命的作用，但鉴于细胞、组织、器官水平衰老进程的复杂性，人们对衰老进程及其机制的了解仍然有限，上述各种延缓衰老的方法和物质的作用效果仍具有不确定性。2016年12月，索尔克研究所的研究人员通过在活体小鼠中高表达Yamanaka四因子(制备诱导多能性干细胞的四因子)的方式，让早衰症小鼠的全身很多器官恢复到相对健康年轻的状态，并延长了小鼠1/3的寿命。因此，用于延缓或者逆转机体大部分细胞或组织器官衰老进程的方法或者物质，如果作用明确，过程可控，有望成为人类抗衰老的可靠技术基础。Although the above substances or methods have the effect of improving the health of the organs and thereby improving the lifespan, in view of the complexity of the aging process of cells, tissues and organs, the understanding of the aging process and its mechanisms is still limited, and the above various anti-aging effects are still limited. The effects of the methods and substances are still uncertain. In December 2016, researchers at the Salk Institute restored many organs of the premature aging mouse to relatively healthy by highly expressing the Yamanaka factor in living mice (preparing the four factors that induce pluripotent stem cells). The young state and extended the life of the mouse 1/3. Therefore, the method or substance used to delay or reverse the aging process of most cells or tissues of the body, if the effect is clear and the process is controllable, is expected to become a reliable technical basis for human anti-aging.
转基因技术虽然在早衰症小鼠身上实现了全身大部分脏器的衰老逆转，但鉴于安全性考虑，此方法在人体内不可行。此外，使用的Yamanaka四因子的激活有致瘤的风险。因此，有必要寻找可用于人体的安全有效的作用方式和物质。小分子化合物或者小分子化合物组合因其具备以下优势：①不导入外源性转录因子，不改变源细胞的基因结构，具有良好的安全性与稳定性；②作用系统较为稳定，易于质控，成本低廉；③作用过程短，效率高，便于规模化生产；④便于人体吸收和代谢，将成为减缓或者逆转衰老进程的有效物质。然而，小分 子化合物或者小分子化合物组合抗衰老的的报道和研究很少，一方面由于缺乏筛选该类小分子化合物的方法和体系；另一方面，即使在小鼠上尝试有用的小分子化合物，由于人类与小鼠存在约25％的基因差异，应用于人的同类细胞，其可行性也不高。因此，有针对性地筛选可安全并明确地延缓或逆转人类衰老进程的小分子化合物，对延缓人类衰老进程、提高健康状态及生活质量具有重要意义。Although transgenic technology has achieved aging reversal of most organs in premature aging mice, this method is not feasible in humans due to safety considerations. In addition, the activation of the four-factor Yamanaka has a risk of tumorigenicity. Therefore, it is necessary to find safe and effective modes of action and substances that can be used in the human body. Small molecule compounds or small molecule compound combinations have the following advantages: 1 no introduction of exogenous transcription factors, no change in the genetic structure of the source cells, good safety and stability; 2 system of action is relatively stable, easy to control, Low cost; 3 short process, high efficiency, easy to scale production; 4 easy for human body absorption and metabolism, will become an effective substance to slow down or reverse the aging process. However, there have been few reports and studies on anti-aging of small molecule compounds or small molecule compounds, on the one hand due to the lack of methods and systems for screening such small molecule compounds; on the other hand, even useful small molecule compounds have been tried on mice. Because humans and mice have about 25% genetic differences, the feasibility of applying them to human cells is not high. Therefore, targeted screening of small molecule compounds that can safely and explicitly delay or reverse the process of human aging is important for delaying the process of human aging, improving health and quality of life.
发明内容Summary of the invention
本发明提供一种用于预防、延缓或逆转细胞、组织、器官、机体衰老的小分子化合物/组合，产品及其用途。所述小分子化合物组合在细胞、组织、器官、机体抗衰老的过程中具有可调控性、整体性、可逆转性和安全性。The present invention provides a small molecule compound/combination, product and use thereof for preventing, delaying or reversing cell, tissue, organ, and body aging. The small molecule compound combination has regulation, integrity, reversibility and safety in the process of anti-aging of cells, tissues, organs and organisms.
本发明第一方面，提供一种预防、延缓或者逆转哺乳动物如人的细胞、组织、器官或机体衰老进程的小分子化合物组合，所述小分子化合物组合包括DNMT抑制剂、HMT抑制剂、组蛋白去甲基化酶抑制剂、TGF-β抑制剂、WNT/β-catenin激动剂、cAMP激动剂和赖氨酸脱乙酰基酶抑制剂中的至少一种。According to a first aspect of the present invention, there is provided a combination of small molecule compounds for preventing, delaying or reversing the aging process of cells, tissues, organs or organisms of a mammal such as a human, the combination of small molecule compounds comprising a DNMT inhibitor, an HMT inhibitor, a group At least one of a protein demethylase inhibitor, a TGF-β inhibitor, a WNT/β-catenin agonist, a cAMP agonist, and a lysine deacetylase inhibitor.
作为选择，所述小分子化合物组合为DNMT抑制剂、HMT抑制剂、组蛋白去甲基化酶抑制剂和赖氨酸脱乙酰基酶抑制剂中的至少一种。Alternatively, the small molecule compound is combined into at least one of a DNMT inhibitor, an HMT inhibitor, a histone demethylase inhibitor, and a lysine deacetylase inhibitor.
作为选择，所述小分子化合物组合为TGF-β抑制剂、WNT/β-catenin激动剂、cAMP激动剂、DNMT抑制剂、HMT抑制剂和赖氨酸脱乙酰基酶抑制剂中的至少一种。Alternatively, the small molecule compound is combined into at least one of a TGF-β inhibitor, a WNT/β-catenin agonist, a cAMP agonist, a DNMT inhibitor, an HMT inhibitor, and a lysine deacetylase inhibitor. .
进一步地，所述小分子化合物还包括RAR激动剂、ascorbate(抗坏血酸)和ROCK抑制剂中的至少一种。Further, the small molecule compound further includes at least one of an RAR agonist, an ascorbate (ascorbic acid), and a ROCK inhibitor.
进一步地，所述小分子化合物组合包括按时序分阶段使用的第一阶段化合物和第二阶段化合物，所述第一阶段化合物为DNMT抑制剂、HMT抑制剂、组蛋白去甲基化酶抑制剂、T GF-β抑制剂、WNT/β-catenin激动剂、cAMP激动剂和赖氨酸脱乙酰基酶抑制剂中的至少一种；Further, the small molecule compound combination comprises a first stage compound and a second stage compound which are used in stages according to a time series, the first stage compound being a DNMT inhibitor, an HMT inhibitor, a histone demethylase inhibitor At least one of a T GF-β inhibitor, a WNT/β-catenin agonist, a cAMP agonist, and a lysine deacetylase inhibitor;
所述第二阶段化合物为DNMT抑制剂、HMT抑制剂、组蛋白去甲基化酶抑制剂、TGF-β抑制剂，WNT/β-catenin激动剂、cAMP激动剂、赖氨酸脱乙酰基酶抑制剂、RAR激动剂、ascorbate(抗坏血酸)和ROCK抑制剂中的至少一种。The second stage compound is a DNMT inhibitor, an HMT inhibitor, a histone demethylase inhibitor, a TGF-β inhibitor, a WNT/β-catenin agonist, a cAMP agonist, a lysine deacetylase At least one of an inhibitor, a RAR agonist, an ascorbate (ascorbic acid), and a ROCK inhibitor.
所述TGF-β受体抑制剂为I型TGF-β受体抑制剂，所述cAMP激动剂为EPAC/RAP1激动剂。The TGF-beta receptor inhibitor is a type I TGF-beta receptor inhibitor and the cAMP agonist is an EPAC/RAP1 agonist.
进一步地，所述小分子化合物组合与PDGF-AB组合使用，提高小分子化合物组合的作用效率。Further, the small molecule compound combination is used in combination with PDGF-AB to improve the efficiency of the action of the small molecule compound combination.
作为优选，所述赖氨酸脱乙酰基酶抑制剂包括sodium phenylbutyrate，butyrate，sodiu m butyrate，MC1568，CI994(Tacedinaline)，chidamide，CAY10683(SantacruzaMate A)，CUD C-907，M344(Histone Deacetylase Inhibitor III)，LAQ824(NVP-LAQ824，Dacinostat)，Prac  inostat(SB939)，VPA(Valproic acid)，Valproic acid sodium salt,Scriptaid，Apicidin，LBH-589(Panobinostat)，MS-275，SAHA(Vorinostat)，Trichostatin(TSA)，Psammaplin A，PCI-24781(A bexinostat)，Rocilinostat(ACY-1215)，Mocetinostat(MGCD0103)，4-Phenylbutyrate(4PB)，sp litomicin，SRT1720，resveratrol，Sirtinol，APHA，CI-994，Depudecin，FK-228，HC-Toxin，ITF-2357(Givinostat)，Chidamide，RGFP 966，PHOB，BG45，Nexturastat A，TMP269，C AY10603，MGCD-0103，Niltubacin，PXD-101(Belinostat)，Pyroxamide，Tubacin，EX-527，BATCP，Cambinol，MOCPAC，PTACH，MC1568，NCH51和TC-H106中的至少一种；Preferably, the lysine deacetylase inhibitor comprises sodium phenylbutyrate, butyrate, sodiu m butyrate, MC1568, CI994 (Tacedinaline), chidamide, CAY10683 (SantacruzaMate A), CUD C-907, M344 (Histone Deacetylase Inhibitor III) ), LAQ824 (NVP-LAQ824, Dacinostat), Prac inostat (SB939), VPA (Valproic acid), Valproic acid sodium salt, Scriptaid, Apicidin, LBH-589 (Panobinostat), MS-275, SAHA (Vorinostat), Trichostatin ( TSA), Psammaplin A, PCI-24781 (A bexinostat), Rocilinostat (ACY-1215), Mocetinostat (MGCD0103), 4-Phenylbutyrate (4PB), sp litomicin, SRT1720, resveratrol, Sirtinol, APHA, CI-994, Depudecin, FK-228, HC-Toxin, ITF-2357 (Givinostat), Chidamide, RGFP 966, PHOB, BG45, Nexturastat A, TMP269, C AY10603, MGCD-0103, Niltubacin, PXD-101 (Belinostat), Pyroxamide, Tubacin, EX At least one of -527, BATCP, Cambinol, MOCPAC, PTACH, MC1568, NCH51 and TC-H106;
所述TGF-β受体抑制剂包括616452，LY2109761，Pirfenidone，Repsox(E-616452)，SB431542，A77-01，Tranilast，Galunisertib(LY2157299)，A8301，GW788388，ITD-1，SD208，S B525334，LY364947，ASP3029，D4476和SB505124中的至少一种；The TGF-beta receptor inhibitors include 616452, LY2109761, Pirfenidone, Repox (E-616452), SB431542, A77-01, Tranilast, Galunisertib (LY2157299), A8301, GW788388, ITD-1, SD208, S B525334, LY364947 At least one of ASP3029, D4476 and SB505124;
所述WNT/β-catenin激动剂包括MAY-262611，CHIR98014，CHIR99021，LiCl，Li 2CO 3，TD114-2，AZD2858，AZD1080，BIO，Kenpaullone，TWS119，LY2090314，CBM1078，SB216763和AR-A014418中的至少一种； The WNT/β-catenin agonists include MAY-262611, CHIR98014, CHIR99021, LiCl, Li 2 CO 3 , TD114-2, AZD2858, AZD1080, BIO, Kenpaullone, TWS119, LY2090314, CBM1078, SB216763 and AR-A014418 At least one
所述cAMP激动剂包括EPAC/RAP1激动剂，8-Bromo-cAMP，Dibutyryl-Camp和Sp-8-Br-cAMPs中的至少一种；The cAMP agonist comprises at least one of an EPAC/RAP1 agonist, 8-Bromo-cAMP, Dibutyryl-Camp and Sp-8-Br-cAMPs;
所述EPAC/RAP1激动剂包括Forskolin，IBMX，Prostaglandin E2(PGE2)，NKH477，8-pCPT-2′-O-Me-cAMP，GSK256066，Apremilast(CC-10004)，Roflumilast，Cilomilast，Rolip ram和Milrinone中的至少一种；The EPAC/RAP1 agonists include Forskolin, IBMX, Prostaglandin E2 (PGE2), NKH477, 8-pCPT-2'-O-Me-cAMP, GSK256066, Apremilast (CC-10004), Roflumilast, Cilomilast, Rolip ram and Milrinone At least one of them;
所述RAR激动剂包括TTNPB，Bexarotene，Ch55，Tamibarotene，Retinol，AM580，AT RA，13-cis RA，Vitamin A及Vitamin A衍生物中的至少一种；The RAR agonist comprises at least one of TTNPB, Bexarotene, Ch55, Tamibarotene, Retinol, AM580, AT RA, 13-cis RA, Vitamin A and Vitamin A derivatives;
所述ROCK抑制剂包括Y-27632，Y-27632 2HCl，Thiazovivin，Ripasudil(K-115)，Fasu dil，Fasudil(HA-1077)HCl，GSK429286A，RKI-1447和PKI-1313中的至少一种；The ROCK inhibitor comprises at least one of Y-27632, Y-27632 2HCl, Thiazovivin, Ripasudil (K-115), Fasu dil, Fasudil (HA-1077) HCl, GSK429286A, RKI-1447 and PKI-1313;
所述DNMT抑制剂包括RG108，Thioguanine，5-Aza-2'-deoxycytidine(Decitabine)，SGI-1027，Zebularine和5-Azacytidine(AZA)中的至少一种；The DNMT inhibitor comprises at least one of RG108, Thioguanine, 5-Aza-2'-deoxycytidine (Decitabine), SGI-1027, Zebularine and 5-Azacytidine (AZA);
所述HMT抑制剂包括EPZ004777，EPZ5676，GSK503，BIX 01294，DZNep，DZNep HCL,SGC 0946中的至少一种；The HMT inhibitor comprises at least one of EPZ004777, EPZ5676, GSK503, BIX 01294, DZNep, DZNep HCL, SGC 0946;
所述组蛋白去甲基化酶抑制剂包括parnate(tranylcypromine)，Tranylcypromine(2-PCPA)HCl SP2509，4SC-202，ORY-1001(RG-6016)，GSKJ1和GSK-LSD1中的至少一种。The histone demethylase inhibitor comprises at least one of parnate (tranylcypromine), Tranylcypromine (2-PCPA) HCl SP2509, 4SC-202, ORY-1001 (RG-6016), GSKJ1 and GSK-LSD1.
作为进一步优选，所述赖氨酸脱乙酰基酶抑制剂为sodium butyrate,VPA和Trichostatin(T SA)的至少一种；Further preferably, the lysine deacetylase inhibitor is at least one of sodium butyrate, VPA and Trichostatin (T SA);
所述TGF-β受体抑制剂为Repsox(E-616452)和SB431542中的至少一种；The TGF-β receptor inhibitor is at least one of Repx (E-616452) and SB431542;
所述WNT/β-catenin激动剂为CHIR99021和BIO中的至少一种；The WNT/β-catenin agonist is at least one of CHIR99021 and BIO;
所述cAMP激动剂为Forskolin和Rolipram中的至少一种；The cAMP agonist is at least one of Forskolin and Rolipram;
所述RAR激动剂为TTNPB和AM580中的至少一种；The RAR agonist is at least one of TTNPB and AM580;
所述ROCK抑制剂包括Y-27632和Thiazovivin中的至少一种；The ROCK inhibitor comprises at least one of Y-27632 and Thiazovivin;
所述DNMT抑制剂为RG108,5-Aza-2'-deoxycytidine(Decitabine)和5-Azacytidine(AZA)中的至少一种；The DNMT inhibitor is at least one of RG108, 5-Aza-2'-deoxycytidine (Decitabine) and 5-Azacytidine (AZA);
所述HMT抑制剂为EPZ004777，EPZ5676和SGC 0946中的至少一种；The HMT inhibitor is at least one of EPZ004777, EPZ5676 and SGC 0946;
所述组蛋白去甲基化酶的抑制剂为parnate(tranylcypromine)和Tranylcypromine(2-PCPA)HCl的至少一种。The inhibitor of the histone demethylase is at least one of parnate (tranylcypromine) and Tranylcypromine (2-PCPA) HCl.
所述小分子化合物组合用于延长哺乳动物如人的细胞的端粒长度，瞬时刺激端粒相关蛋白的表达或上调，如TERT(端粒酶反转录酶)的表达和TPP1上调；作用后不会刺激哺乳动物如人的细胞表达干性基因，如OCT4，Nanog等。The combination of small molecule compounds is used to prolong the telomere length of mammalian cells such as humans, transiently stimulate the expression or up-regulation of telomere-associated proteins, such as TERT (telomerase reverse transcriptase) expression and TPP1 up-regulation; It does not stimulate mammalian cells such as humans to express dry genes such as OCT4, Nanog, and the like.
本发明的一些实施方案，具体小分子化合物的有效浓度如下，以下给出的浓度范围只是参考，可在此基础上做适应性修改，如果其他小分子替代以下小分子，浓度也可以做适应性调整。Some embodiments of the present invention, the effective concentration of the specific small molecule compound is as follows, the concentration range given below is only a reference, and can be adapted on this basis, if other small molecules replace the following small molecules, the concentration can also be adapted. Adjustment.
Forskolin浓度为2μM～20μM；Repsox浓度为2～15uM；CHIR99021浓度为1μM～10μM；VPA浓度为0.5mM～1.5mM；TTNPB浓度为3μM～8μM；AM580浓度为0.03～0.08μM；EPZ004777浓度为3～8μM；Y-27632浓度为3～15μM，L-Ascorbin acid 2-phosphate浓度为0.15～0.25mM；5-Aza-2'-deoxycytidine浓度为0.5～20μM。Forskolin concentration is 2μM～20μM; Repsox concentration is 2～15uM; CHIR99021 concentration is 1μM～10μM; VPA concentration is 0.5mM～1.5mM; TTNPB concentration is 3μM～8μM; AM580 concentration is 0.03～0.08μM; EPZ004777 concentration is 3～ 8 μM; Y-27632 concentration is 3-15 μM, L-Ascorbin acid 2-phosphate concentration is 0.15-0.25 mM; 5-Aza-2'-deoxycytidine concentration is 0.5-20 μM.
本发明预防、延缓或逆转衰老的机理如下：本发明通过对老化或者衰老的细胞进行表观遗传学上的调节，或者通过激活早期发育相关的转录因子，如激活WNT/β-catenin信号通路、激活cAMP信号通路、激动RA信号通路，抑制TGF-beta信号通路,实现细胞衰老的预防，延缓或者逆转。所述小分子化合物组合可用于延长哺乳动物如人的细胞的端粒长度，瞬时刺激端粒相关蛋白的表达/上调，如TERT(端粒酶反转录酶)和TPP1，不会刺激哺乳动物如人的细胞表达干性基因，如OCT4，Nanog等，安全有效。The mechanism of the present invention for preventing, delaying or reversing aging is as follows: the present invention by epigenetical regulation of aging or senescent cells, or by activating early development-associated transcription factors, such as activation of the WNT/β-catenin signaling pathway, Activation of cAMP signaling pathway, agonistic RA signaling pathway, inhibition of TGF-beta signaling pathway, prevention, delay or reversal of cellular senescence. The combination of small molecule compounds can be used to prolong telomere length in mammalian cells such as humans, transiently stimulate expression/upregulation of telomere-associated proteins, such as TERT (telomerase reverse transcriptase) and TPP1, without stimulating mammals For example, human cells express dry genes, such as OCT4, Nanog, etc., safe and effective.
本发明第二方面，提供一种包含所述小分子化合物组合的试剂盒、培养液/基、药物、保健品、食品、化妆品或医疗器械。According to a second aspect of the present invention, a kit, a culture solution/base, a medicine, a health care product, a food, a cosmetic or a medical device comprising the combination of the small molecule compounds is provided.
本发明第三方面，提供一种所述小分子化合物组合在预防、延缓或者逆转哺乳动物如人的细胞、组织、器官或机体衰老进程和相关疾病中的应用及提高哺乳动物如人组织、器官修复能力的应用。According to a third aspect of the present invention, a use of the small molecule compound combination for preventing, delaying or reversing a cell, tissue, organ or body aging process and related diseases of a mammal such as a human, and for improving a mammal such as a human tissue or an organ The application of repair capabilities.
所述细胞来源于哺乳动物如人的机体细胞或体外培养的细胞。The cells are derived from a mammalian cell such as a human body or a cell cultured in vitro.
本发明第四方面，提供一种由所述小分子化合物组合预防、延缓或者逆转哺乳动物如人的细胞、组织、器官或机体衰老进程的方法。According to a fourth aspect of the present invention, a method for preventing, delaying or reversing the aging process of a cell, tissue, organ or body of a mammal such as a human by the combination of the small molecule compound is provided.
本发明第五方面，提供一种通过所述小分子化合物组合作用得到的年轻化的细胞及细胞产物。According to a fifth aspect of the present invention, a younger cell and a cell product obtained by a combination of the small molecule compounds are provided.
本发明第六方面，提供一种所述年轻化的细胞及细胞产物的应用。In a sixth aspect of the invention, there is provided the use of the youngened cells and cell products.
本说明书中采用的术语“衰老”与“老化”的意思相同,年轻化的细胞指的是衰老前的细 胞。但本发明中所述的“衰老”不包含永久停止分裂的细胞。The term "aging" as used in this specification has the same meaning as "aging", and younger cells refer to cells before aging. However, the "aging" described in the present invention does not include cells that permanently stop dividing.
本发明具有以下有益效果：本发明采用小分子化合物或其组合预防、延缓或逆转细胞、组织、器官、机体衰老，延长细胞端粒长度，作用靶点明确、成份清楚、成熟稳定、可准确调控抗衰老的进程；并且小分子化合物易于质控及规模化生产。由于在作用过程中机体或细胞不表达干性基因，安全性高；而且小分子化合物及其组合对机体易于吸收，能够分别在细胞、组织、器官、机体的整体水平上，预防、延缓和逆转老化，对延缓人类衰老进程、提高健康状态及生活质量具有重要意义。The invention has the following beneficial effects: the invention adopts a small molecule compound or a combination thereof to prevent, delay or reverse the aging of cells, tissues, organs and organisms, prolong the telomere length of the cells, and has a clear target, clear composition, stable and stable, and can be accurately regulated. Anti-aging process; and small molecule compounds are easy to control and scale production. Because the body or cells do not express dry genes during the process, the safety is high; and the small molecule compounds and their combinations are easy to absorb, which can prevent, delay and reverse the cells, tissues, organs and organisms. Aging is of great significance in delaying the process of human aging, improving health and quality of life.
附图说明DRAWINGS
图1为小分子化合物作用后人源皮肤成纤维细胞形态图；Figure 1 is a view showing the morphology of human skin fibroblasts after the action of small molecule compounds;
图2为小分子化合物作用后人源骨髓间充质干细胞形态图；2 is a morphology diagram of human bone marrow mesenchymal stem cells after treatment with a small molecule compound;
图3为小分子化合物作用后人源皮肤成纤维细胞细胞性状的鉴定；Figure 3 is a diagram showing the identification of human dermal fibroblast cell traits after the action of small molecule compounds;
图4为小分子化合物作用后人源骨髓间充质干细胞细胞性状的鉴定；Figure 4 shows the identification of cell traits of human bone marrow mesenchymal stem cells after treatment with small molecule compounds;
图5为通过小分子化合物作用后人源细胞的端粒长度变化；Figure 5 is a graph showing changes in telomere length of human cells after treatment with small molecule compounds;
图6为通过小分子化合物作用后人源细胞的端粒相关基因的表达；Figure 6 shows the expression of telomere-associated genes in human cells after treatment with small molecule compounds;
图7为对小分子化合物作用后人源皮肤成纤维细胞中与衰老相关基因的表达变化图；Figure 7 is a graph showing changes in expression of aging-related genes in human dermal fibroblasts after treatment with small molecule compounds;
图8为小分子化合物作用后人源皮肤成纤维细胞体外功能活力的检测结果图；Figure 8 is a graph showing the results of in vitro functional viability of human skin fibroblasts after action of small molecule compounds;
图9为小分子化合物作用后人源皮肤成纤维细胞体内功能活力的检测结果图；Figure 9 is a graph showing the results of in vivo functional activity of human skin fibroblasts after action of small molecule compounds;
图10为对小分子化合物作用后长期传代的细胞干性的检测和成瘤性检测结果图；Figure 10 is a graph showing the results of detection and tumorigenicity of cell dryness after long-term passage of a small molecule compound;
图11为长期传代的小分子化合物作用后人源皮肤成纤维细胞的克隆形成及形态图。Figure 11 is a clonal formation and morphological map of human dermal fibroblasts after long-term passage of small molecule compounds.
具体实施方式detailed description
下面结合附图和具体实施例对本发明的技术方案做进一步详细说明，但本发明并不局限于以下技术方案。The technical solutions of the present invention are further described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solutions.
实施例1Example 1
1、皮肤成纤维细胞的分离1. Separation of skin fibroblasts
1.1从志愿者身上获取直径约1cm的皮肤组织块，贴壁法分离皮肤成纤维细胞，分离的细胞培养于基础培养液里，所述基础培养液：10％胎牛血清(Hyclone)+100U/ml青霉素(Sigm a)+100μg/ml链霉素(Sigma)+高糖DMDM。1.1 Obtaining a skin tissue block of about 1 cm in diameter from a volunteer, separating the skin fibroblasts by adherence method, and separating the cells into a basic culture solution: 10% fetal bovine serum (Hyclone) + 100 U/ Ml penicillin (Sigm a) + 100 μg / ml streptomycin (Sigma) + high sugar DMDM.
1.2细胞传代大量扩增，细胞代数在本实验中，细胞为第12代,处理的前一天(Day-1)，接种细胞密度1～2.5×10 4/cm 2培养于37℃，5％CO 2的培养箱中。 1.2 Cell abundance amplification, cell algebra In this experiment, the cell is the 12th generation, the day before treatment (Day-1), the inoculated cell density is 1～2.5×10 4 /cm 2 and cultured at 37 ° C, 5% CO 2 in the incubator.
2、皮肤成纤维细胞的小分子化合物处理2. Treatment of small molecular compounds of skin fibroblasts
更换基础培养液为小分子处理液，培养细胞2-20天，小分子处理液是指：10％胎牛血清(H  yclone)+100U/ml青霉素(Sigma)+100μg/ml链霉素(Sigma)+高糖DMDM培养基(Gibco)+f orskolin(2μM～25μM)+Repsox(2～15uM)+CHIR99021(1μM～10μM)+VPA(0.5mM～1.5mM)+5-Aza-2'-deoxycytidine(0.5～20μM)，该培养系统中10％胎牛血清也可以被血清替代品(invitrogen)以10％～20％的浓度替代；100U/ml青霉素(Sigma)和100μg/ml链霉素(Sig ma)可以不使用。在37℃，5％CO 2环境下培养细胞。 Replace the basal medium as a small molecule treatment solution, culture the cells for 2-20 days, and the small molecule treatment solution means: 10% fetal bovine serum (H yclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma + high glucose DMDM medium (Gibco) + f orskolin (2μM ~ 25μM) + Repx (2 ~ 15uM) + CHIR99021 (1μM ~ 10μM) + VPA (0.5mM ~ 1.5mM) + 5-Aza-2'-deoxycytidine (0.5-20 μM), 10% fetal bovine serum in this culture system can also be replaced by serum replacement (invitrogen) at a concentration of 10% to 20%; 100 U/ml penicillin (Sigma) and 100 μg/ml streptomycin (Sig) Ma) can not be used. The cells were cultured at 37 ° C in a 5% CO 2 atmosphere.
3、皮肤成纤维细胞的维持培养3. Maintenance culture of skin fibroblasts
上述第2步骤的处理结束后，完全更换为基础培养液，正常维持培养，在37℃，5％CO 2环境下培养细胞。所述基础培养液：10％胎牛血清(Hyclone)+100U/ml青霉素(Sigma)+100μg/ml链霉素(Sigma)+高糖DMDM,中10％胎牛血清也可以被血清替代品(invitrogen)以10％～20％的浓度替代；100U/ml青霉素(Sigma)和100μg/ml链霉素(Sigma)可以不使用。 After the completion of the treatment in the second step described above, the basal culture solution was completely replaced, the culture was maintained normally, and the cells were cultured at 37 ° C in a 5% CO 2 atmosphere. The basal medium is: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high sugar DMDM, 10% fetal bovine serum can also be replaced by serum ( Invitrogen) is replaced by a concentration of 10% to 20%; 100 U/ml penicillin (Sigma) and 100 μg/ml streptomycin (Sigma) may not be used.
4、处理后细胞性状的鉴定4. Identification of cell traits after treatment
4.1细胞传代到第14代时,使用碧云天细胞衰老β-半乳糖苷酶染色试剂盒进行染色鉴定；4.1 When cells were passaged to the 14th generation, staining was performed using the Biyuntian cell senescence β-galactosidase staining kit;
4.2细胞传代到第14代,使用ebioscience的CD29-PE流式抗体进行染色，对照为同一厂家对应的同型对照品，按照厂家说明书操作，在BD JAZZ流式仪上进行分析；4.2 The cells were passaged to the 14th generation, and stained with e. coli CD29-PE flow antibody. The control was the same type of reference substance of the same manufacturer, and the analysis was performed on the BD JAZZ flow meter according to the manufacturer's instructions.
4.3细胞传代到第14代，在24孔板里培养，使用4％的PFA固定细胞10分钟后，按照免疫荧光的操作步骤，使用santa cruz的Vimentin抗体进行染色；4.3 The cells were passaged to the 14th passage, cultured in a 24-well plate, and the cells were fixed with 4% PFA for 10 minutes, and stained with the Vimentin antibody of Santa cruz according to the procedure of immunofluorescence;
4.4细胞增值的检测，以1.5x10 5作为起始接种细胞量，连续12天计数细胞，每个时间点重复3组； 4.4 Detection of cell proliferation, the amount of cells was inoculated with 1.5 × 10 5 as the initial, the cells were counted for 12 consecutive days, and 3 groups were repeated at each time point;
4.5细胞传代到第14代，收取DNA样本，使用Cawthon qPCR方法进行端粒长度的检测，荧光染料采用Takara SYBR Green I，4.5 cells were passaged to the 14th generation, DNA samples were collected, and the telomere length was detected by the Cawthon qPCR method. The fluorescent dye was Takara SYBR Green I.
端粒扩增引物序列为Telomere amplification primer sequence is
mTel:CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT；mTel: CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT;
mTel:GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT；mTel: GGTCTGCCTACCCCTTACCCTTACCCTTACCCTTACCCT;
单拷贝基因引物Single copy gene primer
36B4-S-F：CAGCAAGTGGGAAGGTGTAATCC；36B4-S-F: CAGCAAGTGGGAAGGTGTAATCC;
36B4-S-R：CCCATTCTATCATCAACGGGTACAA；36B4-S-R: CCCATTCTATCATCAACGGGTACAA;
4.6细胞传代到第14代，收取RNA样本，采用PCR方法进行TERT的检测，人源TERT的引物序列为F：5'-CGGAAGAGTGTCTGGAGCAA-3；R5'-GGATGAAGCGGAGTCTGGA-3'；4.6 cells were passaged to the 14th generation, RNA samples were taken, and TERT was detected by PCR. The primer sequence of human TERT was F: 5'-CGGAAGAGTGTCTGGAGCAA-3; R5'-GGATGAAGCGGAGTCTGGA-3';
4.7细胞传代到第14代，收取RNA样本，通过转录组的分析，定量基因TPP1、CDKN1A、ATF3、GADD45B、BTG2、H2AFX、ITGA6、COL1A1表达量的变化；4.7 cells were passaged to the 14th generation, RNA samples were collected, and the expression levels of genes TPP1, CDKN1A, ATF3, GADD45B, BTG2, H2AFX, ITGA6, COL1A1 were quantified by transcriptome analysis;
4.8异体皮肤移植，检测小分子化合物处理后的人源皮肤成纤维细胞培养液的免疫调节作用。具体方法为：实验组为小分子化合物处理细胞组，未处理细胞组，PBS组，其中传代到第14代，按照动物伦理委员的要求执行动物实验，将供体C57B/6小鼠麻醉,仰卧位固定，全背部脱毛，背部取1.5cm x 1.5cm的全层的皮肤，去除皮下脂肪及血管，将小鼠皮肤剪成直径为1.0cm大小的皮片，置含有无菌PBS的平皿中备用。将剪好的皮片翻转过来放入有生理盐水的培养皿中，用手术刀片轻轻地切去皮下组织至真皮，然后放在无菌生理盐水中备用。4.8 Allogeneic skin transplantation, detection of immunomodulatory effects of human skin fibroblast culture fluid after treatment with small molecule compounds. The specific method is as follows: the experimental group is a small molecule compound treatment cell group, the untreated cell group, the PBS group, which is passaged to the 14th generation, and the animal experiment is performed according to the requirements of the animal ethics committee, and the donor C57B/6 mouse is anesthetized, supine Fixed, full-back hair removal, 1.5cm x 1.5cm full-thickness skin on the back, remove subcutaneous fat and blood vessels, cut the skin of the mouse into a 1.0cm-diameter skin, and place it in a dish containing sterile PBS. . The cut piece of skin was turned over and placed in a petri dish with physiological saline, and the subcutaneous tissue was gently cut into the dermis with a surgical blade, and then placed in sterile physiological saline for use.
受体为BALB/c小鼠，麻醉后背部脱毛，碘酒消毒背部皮肤，制备一直径1.0cm的皮肤缺口，保留血管，于背部逆毛方向移植C57B/6皮片，表面敷盖凡士林油纱和创可贴，松紧适度。移植后1小时内，从尾静脉注射列表中的各组别液体。The recipient was BALB/c mice, the back was depilated after anesthesia, the iodine disinfected the back skin, a skin gap of 1.0 cm in diameter was prepared, the blood vessels were preserved, and the C57B/6 skin was transplanted in the reverse hair direction, and the surface was covered with Vaseline oil yarn. And band-aid, moderately tight. Each group of liquids in the list was injected from the tail vein within 1 hour after transplantation.
4.9细胞克隆形成率的检测：细胞传代到第14代，取对数生长期的细胞，以10个细胞/cm2铺板，每组设3复孔，每隔2天换液，1周～2周左右Giemsa或者PI染色，显微镜下计数形成的克隆数量。4.9 Detection of cell clone formation rate: The cells were passaged to the 14th generation, and the cells in the logarithmic growth phase were plated at 10 cells/cm2. Each group was set with 3 replicate wells, and the solution was changed every 2 days, 1 week to 2 weeks. The number of clones formed was counted under the microscope by Giemsa or PI staining.
实施例2Example 2
1、骨髓间充质干细胞的培养1. Culture of bone marrow mesenchymal stem cells
1.1购买商品化的人源骨髓间充质干细胞或者从骨创伤后志愿者骨碎片残留的骨髓中分离间充质干细胞，细胞培养于基础培养液里，所述基础培养液：8％胎牛血清(Hyclone)+100U/ml青霉素(Sigma)+100μg/ml链霉素(Sigma)+低糖DMDM。1.1 Purchasing commercial human bone marrow mesenchymal stem cells or separating mesenchymal stem cells from bone marrow residual bone fragments after bone trauma, the cells are cultured in a basic culture medium: 8% fetal bovine serum (Hyclone) + 100 U/ml penicillin (Sigma) + 100 μg/ml streptomycin (Sigma) + low sugar DMDM.
1.2细胞传代大量扩增，细胞代数在本实验中，细胞为第10代,处理的前一天(Day-1)，接种细胞密度1～2.5×10 4/cm 2培养于37℃，5％CO 2的培养箱中。 1.2 Cell abduction amplification, cell algebra In this experiment, the cell is the 10th generation, the day before treatment (Day-1), the inoculated cell density is 1~2.5×10 4 /cm 2 and cultured at 37 ° C, 5% CO 2 in the incubator.
2、骨髓间充质干细胞的小分子化合物处理2. Treatment of small molecule compounds of bone marrow mesenchymal stem cells
更换基础培养液为小分子处理液，培养细胞2-20天，小分子处理液是指：10％胎牛血清(Hyclone)+100U/ml青霉素(Sigma)+100μg/ml链霉素(Sigma)+高糖DMDM培养基(Gibco)+forskolin(2μM～25μM)+Repsox(2～15uM)+CHIR99021(1μM～10μM)+VPA(0.5mM～1.5mM)，该培养系统中10％胎牛血清也可以被血清替代品(invitrogen)以10％～20％的浓度替代；100U/ml青霉素(Sigma)和100μg/ml链霉素(Sigma)可以不使用。在37℃，5％CO 2环境下培养细胞。 Replace the basal medium as a small molecule treatment solution, culture the cells for 2-20 days, and the small molecule treatment solution means: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high glucose DMDM medium (Gibco) + forskolin (2μM ~ 25μM) + Repsox (2 ~ 15uM) + CHIR99021 (1μM ~ 10μM) + VPA (0.5mM ~ 1.5mM), 10% fetal bovine serum in the culture system It can be replaced by a serum substitute (invitrogen) at a concentration of 10% to 20%; 100 U/ml penicillin (Sigma) and 100 μg/ml streptomycin (Sigma) may not be used. The cells were cultured at 37 ° C in a 5% CO 2 atmosphere.
3、骨髓间充质干细胞的维持培养3. Maintenance and culture of bone marrow mesenchymal stem cells
上述第2步骤的处理结束后，完全更换为基础培养液，正常维持培养，在37℃，5％CO 2环境下培养细胞。所述基础培养液：8％胎牛血清(Hyclone)+100U/ml青霉素(Sigma)+100μg/ml链霉素(Sigma)+低糖DMDM,中10％胎牛血清也可以被血清替代品(invitrogen)以10％～20％的浓度替代；100U/ml青霉素(Sigma)和100μg/ml链霉素(Sigma)可以不使用。 After the completion of the treatment in the second step described above, the basal culture solution was completely replaced, the culture was maintained normally, and the cells were cultured at 37 ° C in a 5% CO 2 atmosphere. The basal medium: 8% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + low sugar DMDM, 10% fetal bovine serum can also be replaced by serum (invitrogen ) is replaced by a concentration of 10% to 20%; 100 U/ml penicillin (Sigma) and 100 μg/ml streptomycin (Sigma) may not be used.
4、处理后细胞性状的鉴定4. Identification of cell traits after treatment
4.1细胞传代到第12代时,使用碧云天细胞衰老β-半乳糖苷酶染色试剂盒进行染色鉴定；4.1 When the cells were passaged to the 12th generation, staining was performed using the Biyuntian cell senescence β-galactosidase staining kit;
4.2细胞传代到第12代,使用ebioscience的CD29-PE，CD90，CD73，CD34，CD45流式抗体进行染色，对照为同一厂家对应的同型对照品，按照厂家说明书操作，在BD JAZZ流式仪上进行分析；4.2 cells were passaged to the 12th generation, stained with CD29-PE, CD90, CD73, CD34, CD45 flow antibody of ebioscience, the control was the same type of reference substance of the same manufacturer, according to the manufacturer's instructions, on the BD JAZZ flowmeter Conduct analysis;
4.3细胞传代到第12代，使用Cyagen Biosciences的成骨和成脂分化诱导液进行诱导，具体步骤按照产品说明书；4.3 cells were passaged to passage 12 and induced using the osteogenesis and adipogenic differentiation induction solution of Cyagen Biosciences, according to the product specification;
4.4细胞增值的检测，以1.5x10 5作为起始接种细胞量，连续12天计数细胞，每个时间点重复3组； 4.4 Detection of cell proliferation, the amount of cells was inoculated with 1.5 × 10 5 as the initial, the cells were counted for 12 consecutive days, and 3 groups were repeated at each time point;
4.5细胞传代到第14代，收取DNA样本，使用Cawthon qPCR方法进行端粒长度的检测，荧光染料采用Takara SYBR Green I，4.5 cells were passaged to the 14th generation, DNA samples were collected, and the telomere length was detected by the Cawthon qPCR method. The fluorescent dye was Takara SYBR Green I.
端粒扩增引物序列为Telomere amplification primer sequence is
mTel:CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT；mTel: CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT;
mTel:GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT；mTel: GGTCTGCCTACCCCTTACCCTTACCCTTACCCTTACCCT;
单拷贝基因引物Single copy gene primer
36B4-S-F：CAGCAAGTGGGAAGGTGTAATCC；36B4-S-F: CAGCAAGTGGGAAGGTGTAATCC;
36B4-S-R：CCCATTCTATCATCAACGGGTACAA；36B4-S-R: CCCATTCTATCATCAACGGGTACAA;
4.6细胞传代到第14代，收取RNA样本，采用PCR方法进行TERT的检测，人源TERT的引物序列为F：5'-CGGAAGAGTGTCTGGAGCAA-3；R5'-GGATGAAGCGGAGTCTGGA-3'；4.6 cells were passaged to the 14th generation, RNA samples were taken, and TERT was detected by PCR. The primer sequence of human TERT was F: 5'-CGGAAGAGTGTCTGGAGCAA-3; R5'-GGATGAAGCGGAGTCTGGA-3';
4.7细胞传代到第14代，收取RNA样本，通过转录组的分析，定量基因TPP1、CDKN1A、ATF3、GADD45B、BTG2、H2AFX表达量的变化；4.7 cells were passaged to the 14th generation, RNA samples were collected, and the expression levels of genes TPP1, CDKN1A, ATF3, GADD45B, BTG2, and H2AFX were quantified by transcriptome analysis;
4.8异体皮肤移植，检测小分子化合物处理后的人源骨髓间充质干细胞培养液的免疫调节作用。具体方法为：实验组为小分子化合物处理细胞组，未处理细胞组，PBS组，其中传代到第14代，按照动物伦理委员的要求执行动物实验，将供体C57B/6小鼠麻醉,仰卧位固定，全背部脱毛，背部取1.5cm x 1.5cm的全层的皮肤，去除皮下脂肪及血管，将小鼠皮肤剪成直径为1.0cm大小的皮片，置含有无菌PBS的平皿中备用。将剪好的皮片翻转过来放入有生理盐水的培养皿中，用手术刀片轻轻地切去皮下组织至真皮，然后放在无菌生理盐水中备用。4.8 Allogeneic skin transplantation, detection of immunomodulatory effects of human bone marrow mesenchymal stem cell culture medium after treatment with small molecule compounds. The specific method is as follows: the experimental group is a small molecule compound treatment cell group, the untreated cell group, the PBS group, which is passaged to the 14th generation, and the animal experiment is performed according to the requirements of the animal ethics committee, and the donor C57B/6 mouse is anesthetized, supine Fixed, full-back hair removal, 1.5cm x 1.5cm full-thickness skin on the back, remove subcutaneous fat and blood vessels, cut the skin of the mouse into a 1.0cm-diameter skin, and place it in a dish containing sterile PBS. . The cut piece of skin was turned over and placed in a petri dish with physiological saline, and the subcutaneous tissue was gently cut into the dermis with a surgical blade, and then placed in sterile physiological saline for use.
受体为BALB/c小鼠，麻醉后背部脱毛，碘酒消毒背部皮肤，制备一直径1.0cm的皮肤缺口，保留血管，于背部逆毛方向移植C57B/6皮片，表面敷盖凡士林油纱和创可贴，松紧适度。移植后1小时内，从尾静脉注射列表中的各组别液体。The recipient was BALB/c mice, the back was depilated after anesthesia, the iodine disinfected the back skin, a skin gap of 1.0 cm in diameter was prepared, the blood vessels were preserved, and the C57B/6 skin was transplanted in the reverse hair direction, and the surface was covered with Vaseline oil yarn. And band-aid, moderately tight. Each group of liquids in the list was injected from the tail vein within 1 hour after transplantation.
4.9细胞克隆形成率的检测：细胞传代到第14代，取对数生长期的细胞，以10个细胞/cm2铺板，每组设3复孔，每隔2天换液，1周～2周左右Giemsa或者PI染色，显微镜下计数形成的克隆数量。4.9 Detection of cell clone formation rate: The cells were passaged to the 14th generation, and the cells in the logarithmic growth phase were plated at 10 cells/cm2. Each group was set with 3 replicate wells, and the solution was changed every 2 days, 1 week to 2 weeks. The number of clones formed was counted under the microscope by Giemsa or PI staining.
实施例3Example 3
1、皮肤成纤维细胞的分离1. Separation of skin fibroblasts
1.1从志愿者身上获取直径约1cm的皮肤组织块，贴壁法分离皮肤成纤维细胞，分离的细胞培养于基础培养液里，所述基础培养液：10％胎牛血清(Hyclone)+100U/ml青霉素(Sigm a)+100μg/ml链霉素(Sigma)+高糖DMDM。1.1 Obtaining a skin tissue block of about 1 cm in diameter from a volunteer, separating the skin fibroblasts by adherence method, and separating the cells into a basic culture solution: 10% fetal bovine serum (Hyclone) + 100 U/ Ml penicillin (Sigm a) + 100 μg / ml streptomycin (Sigma) + high sugar DMDM.
1.2细胞传代大量扩增，细胞代数在本实验中，细胞为第12代,处理的前一天(Day-1)，接种细胞密度1～2.5×10 4/cm 2培养于37℃，5％CO 2的培养箱中。 1.2 Cell abundance amplification, cell algebra In this experiment, the cell is the 12th generation, the day before treatment (Day-1), the inoculated cell density is 1～2.5×10 4 /cm 2 and cultured at 37 ° C, 5% CO 2 in the incubator.
2、皮肤成纤维细胞的小分子化合物处理2. Treatment of small molecular compounds of skin fibroblasts
更换基础培养液为小分子处理液，培养细胞2-20天，小分子处理液是指：10％胎牛血清(H yclone)+100U/ml青霉素(Sigma)+100μg/ml链霉素(Sigma)+高糖DMDM培养基(Gibco)+f orskolin(2μM～25μM)+5-Aza-2'-deoxycytidine(0.5～20μM)，该培养系统中10％胎牛血清 也可以被血清替代品(invitrogen)以10％～20％的浓度替代；100U/ml青霉素(Sigma)和100μg/ml链霉素(Sigma)可以不使用。在37℃，5％CO 2环境下培养细胞。 Replace the basal medium as a small molecule treatment solution, culture the cells for 2-20 days, and the small molecule treatment solution means: 10% fetal bovine serum (H yclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma + high glucose DMDM medium (Gibco) + f orskolin (2μM ~ 25μM) + 5-Aza-2'-deoxycytidine (0.5 ~ 20μM), 10% fetal bovine serum in this culture system can also be replaced by serum (invitrogen ) is replaced by a concentration of 10% to 20%; 100 U/ml penicillin (Sigma) and 100 μg/ml streptomycin (Sigma) may not be used. The cells were cultured at 37 ° C in a 5% CO 2 atmosphere.
3、皮肤成纤维细胞的维持培养3. Maintenance culture of skin fibroblasts
上述第2步骤的处理结束后，完全更换为基础培养液，正常维持培养，在37℃，5％CO 2环境下培养细胞。所述基础培养液：10％胎牛血清(Hyclone)+100U/ml青霉素(Sigma)+100μg/ml链霉素(Sigma)+高糖DMDM,中10％胎牛血清也可以被血清替代品(invitrogen)以10％～20％的浓度替代；100U/ml青霉素(Sigma)和100μg/ml链霉素(Sigma)可以不使用。 After the completion of the treatment in the second step described above, the basal culture solution was completely replaced, the culture was maintained normally, and the cells were cultured at 37 ° C in a 5% CO 2 atmosphere. The basal medium is: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high sugar DMDM, 10% fetal bovine serum can also be replaced by serum ( Invitrogen) is replaced by a concentration of 10% to 20%; 100 U/ml penicillin (Sigma) and 100 μg/ml streptomycin (Sigma) may not be used.
4、处理后细胞性状的鉴定4. Identification of cell traits after treatment
详细步骤同实施例1Detailed steps are the same as in the first embodiment
实施例4Example 4
1、皮肤成纤维细胞的分离1. Separation of skin fibroblasts
1.1从志愿者身上获取直径约1cm的皮肤组织块，贴壁法分离皮肤成纤维细胞，分离的细胞培养于基础培养液里，所述基础培养液：10％胎牛血清(Hyclone)+100U/ml青霉素(Sigm a)+100μg/ml链霉素(Sigma)+高糖DMDM。1.1 Obtaining a skin tissue block of about 1 cm in diameter from a volunteer, separating the skin fibroblasts by adherence method, and separating the cells into a basic culture solution: 10% fetal bovine serum (Hyclone) + 100 U/ Ml penicillin (Sigm a) + 100 μg / ml streptomycin (Sigma) + high sugar DMDM.
1.2细胞传代大量扩增，细胞代数在本实验中，细胞为第12代,处理的前一天(Day-1)，接种细胞密度1～2.5×10 4/cm 2培养于37℃，5％CO 2的培养箱中。 1.2 Cell abundance amplification, cell algebra In this experiment, the cell is the 12th generation, the day before treatment (Day-1), the inoculated cell density is 1～2.5×10 4 /cm 2 and cultured at 37 ° C, 5% CO 2 in the incubator.
2、皮肤成纤维细胞的小分子化合物处理2. Treatment of small molecular compounds of skin fibroblasts
更换基础培养液为小分子处理液，培养细胞2-20天，小分子处理液是指：10％胎牛血清(H yclone)+100U/ml青霉素(Sigma)+100μg/ml链霉素(Sigma)+高糖DMDM培养基(Gibco)+V PA(0.5mM～1.5mM)+Repsox(2～15uM)，该培养系统中10％胎牛血清也可以被血清替代品(invitrogen)以10％～20％的浓度替代；100U/ml青霉素(Sigma)和100μg/ml链霉素(Sig ma)可以不使用。在37℃，5％CO 2环境下培养细胞。 Replace the basal medium as a small molecule treatment solution, culture the cells for 2-20 days, and the small molecule treatment solution means: 10% fetal bovine serum (H yclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma + high glucose DMDM medium (Gibco) + V PA (0.5 mM ~ 1.5 mM) + Repx (2 ~ 15 uM), 10% fetal bovine serum in this culture system can also be 10% ~ by serum substitute (invitrogen) 20% concentration substitution; 100 U/ml penicillin (Sigma) and 100 μg/ml streptomycin (Sig ma) may not be used. The cells were cultured at 37 ° C in a 5% CO 2 atmosphere.
3、皮肤成纤维细胞的维持培养3. Maintenance culture of skin fibroblasts
上述第2步骤的处理结束后，完全更换为基础培养液，正常维持培养，在37℃，5％CO 2环境下培养细胞。所述基础培养液：10％胎牛血清(Hyclone)+100U/ml青霉素(Sigma)+100μg/ml链霉素(Sigma)+高糖DMDM,中10％胎牛血清也可以被血清替代品(invitrogen)以10％～20％的浓度替代；100U/ml青霉素(Sigma)和100μg/ml链霉素(Sigma)可以不使用。 After the completion of the treatment in the second step described above, the basal culture solution was completely replaced, the culture was maintained normally, and the cells were cultured at 37 ° C in a 5% CO 2 atmosphere. The basal medium is: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high sugar DMDM, 10% fetal bovine serum can also be replaced by serum ( Invitrogen) is replaced by a concentration of 10% to 20%; 100 U/ml penicillin (Sigma) and 100 μg/ml streptomycin (Sigma) may not be used.
4、处理后细胞性状的鉴定4. Identification of cell traits after treatment
详细步骤同实施例1。The detailed steps are the same as in the first embodiment.
实施例5Example 5
第2步骤中皮肤成纤维细胞的小分子化合物处理为：更换基础培养液为小分子处理液，培养细胞2-20天，小分子处理液是指：10％胎牛血清(Hyclone)+100U/ml青霉素(Sigma)+100μg/ml链霉素(Sigma)+高糖DMDM培养基(Gibco)+VPA(0.5mM～1.5mM)+Repsox(2～15u M)+5-Aza-2'-deoxycytidine(0.5～20μM),该培养系统中10％胎牛血清也可以被血清替代品(in  vitrogen)以10％～20％的浓度替代；100U/ml青霉素(Sigma)和100μg/ml链霉素(Sigma)可以不使用。在37℃，5％CO 2环境下培养细胞。其余步骤同实施例1。 In the second step, the small molecule compound of the skin fibroblasts is treated as follows: the base culture solution is replaced with a small molecule treatment solution, and the cells are cultured for 2-20 days, and the small molecule treatment solution means: 10% fetal bovine serum (Hyclone) + 100 U/ Ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high glucose DMDM medium (Gibco) + VPA (0.5 mM ~ 1.5 mM) + Repx (2 ~ 15u M) + 5-Aza-2'-deoxycytidine (0.5-20 μM), 10% fetal bovine serum in this culture system can also be replaced by serum substitute (in vitrogen) at a concentration of 10% to 20%; 100 U/ml penicillin (Sigma) and 100 μg/ml streptomycin ( Sigma) can not be used. Cells were cultured at 37 ℃, 5% CO 2 environment. The remaining steps are the same as in the first embodiment.
实施例6Example 6
第2步骤中皮肤成纤维细胞的小分子化合物处理为：更换基础培养液为小分子处理液，培养细胞2-20天，小分子处理液是指：10％胎牛血清(Hyclone)+100U/ml青霉素(Sigma)+100μg/ml链霉素(Sigma)+高糖DMDM培养基(Gibco)+VPA(0.5mM～3mM)，该培养系统中10％胎牛血清也可以被血清替代品(invitrogen)以10％～20％的浓度替代；100U/ml青霉素(Sigm a)和100μg/ml链霉素(Sigma)可以不使用。在37℃，5％CO 2环境下培养细胞。其余步骤同实施例1。 In the second step, the small molecule compound of the skin fibroblasts is treated as follows: the base culture solution is replaced with a small molecule treatment solution, and the cells are cultured for 2-20 days, and the small molecule treatment solution means: 10% fetal bovine serum (Hyclone) + 100 U/ Ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high glucose DMDM medium (Gibco) + VPA (0.5 mM ~ 3 mM), 10% fetal bovine serum in this culture system can also be replaced by serum (invitrogen ) is replaced by a concentration of 10% to 20%; 100 U/ml penicillin (Sigm a) and 100 μg/ml streptomycin (Sigma) may not be used. The cells were cultured at 37 ° C in a 5% CO 2 atmosphere. The remaining steps are the same as in the first embodiment.
实施例7Example 7
第2步骤中皮肤成纤维细胞的小分子化合物处理为：更换基础培养液为小分子处理液，培养细胞2-20天，小分子处理液是指：10％胎牛血清(Hyclone)+100U/ml青霉素(Sigma)+100μg/ml链霉素(Sigma)+高糖DMDM培养基(Gibco)+5-Aza-2'-deoxycytidine(0.2～20μM)，该培养系统中10％胎牛血清也可以被血清替代品(invitrogen)以10％～20％的浓度替代；100U/ml青霉素(Sigma)和100μg/ml链霉素(Sigma)可以不使用。在37℃，5％CO 2环境下培养细胞。其余步骤同实施例1。 In the second step, the small molecule compound of the skin fibroblasts is treated as follows: the base culture solution is replaced with a small molecule treatment solution, and the cells are cultured for 2-20 days, and the small molecule treatment solution means: 10% fetal bovine serum (Hyclone) + 100 U/ Ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high glucose DMDM medium (Gibco) + 5-Aza-2'-deoxycytidine (0.2 ~ 20 μM), 10% fetal bovine serum can also be used in the culture system It is replaced by a serum substitute (invitrogen) at a concentration of 10% to 20%; 100 U/ml penicillin (Sigma) and 100 μg/ml streptomycin (Sigma) may not be used. The cells were cultured at 37 ° C in a 5% CO 2 atmosphere. The remaining steps are the same as in the first embodiment.
实施例8Example 8
第2步骤中皮肤成纤维细胞的小分子化合物处理为：更换基础培养液为小分子处理液，培养细胞2-20天，小分子处理液是指：10％胎牛血清(Hyclone)+100U/ml青霉素(Sigma)+100μg/ml链霉素(Sigma)+高糖DMDM培养基(Gibco)+RG108(0.5～30μM)，该培养系统中10％胎牛血清也可以被血清替代品(invitrogen)以10％～20％的浓度替代；100U/ml青霉素(Sigma)和100μg/ml链霉素(Sigma)可以不使用。在37℃，5％CO 2环境下培养细胞。其余步骤同实施例1。 In the second step, the small molecule compound of the skin fibroblasts is treated as follows: the base culture solution is replaced with a small molecule treatment solution, and the cells are cultured for 2-20 days, and the small molecule treatment solution means: 10% fetal bovine serum (Hyclone) + 100 U/ Ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high sugar DMDM medium (Gibco) + RG108 (0.5 ~ 30 μM), 10% fetal bovine serum in this culture system can also be replaced by serum (invitrogen) Replaced at a concentration of 10% to 20%; 100 U/ml penicillin (Sigma) and 100 μg/ml streptomycin (Sigma) may not be used. The cells were cultured at 37 ° C in a 5% CO 2 atmosphere. The remaining steps are the same as in the first embodiment.
实施例9Example 9
第2步骤中皮肤成纤维细胞的小分子化合物处理为：更换基础培养液为小分子处理液，培养细胞2-20天，小分子处理液是指：10％胎牛血清(Hyclone)+100U/ml青霉素(Sigma)+100μg/ml链霉素(Sigma)+高糖DMDM培养基(Gibco)+Repsox(2～15uM)+CHIR99021(1μM～10μM)+VPA(0.5mM～1.5mM)+TTNPB(3μM～8μM)+AM580(0.03～0.08μM)+EPZ004777(3～8μM)+Y-27632(3～15μM)+L-Ascorbin acid 2-phosphate(0.15～0.25m M)，该培养系统中10％胎牛血清也可以被血清替代品(invitrogen)以10％～20％的浓度替代；100U/ml青霉素(Sigma)和100μg/ml链霉素(Sigma)可以不使用。在37℃，5％CO 2环境下培养细胞。其余步骤同实施例1。 In the second step, the small molecule compound of the skin fibroblasts is treated as follows: the base culture solution is replaced with a small molecule treatment solution, and the cells are cultured for 2-20 days, and the small molecule treatment solution means: 10% fetal bovine serum (Hyclone) + 100 U/ Ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high glucose DMDM medium (Gibco) + Repsox (2 ~ 15 uM) + CHIR99021 (1 μM ~ 10 μM) + VPA (0.5 mM ~ 1.5 mM) + TTNPB ( 3μM～8μM)+AM580(0.03～0.08μM)+EPZ004777(3～8μM)+Y-27632(3～15μM)+L-Ascorbin acid 2-phosphate(0.15～0.25m M), 10% in the culture system Fetal bovine serum can also be replaced by a serum replacement (invitrogen) at a concentration of 10% to 20%; 100 U/ml penicillin (Sigma) and 100 μg/ml streptomycin (Sigma) may not be used. The cells were cultured at 37 ° C in a 5% CO 2 atmosphere. The remaining steps are the same as in the first embodiment.
实施例10Example 10
第2步骤中皮肤成纤维细胞的小分子化合物处理为：更换基础培养液为小分子处理液， 培养细胞2-20天，小分子处理液是指：10％胎牛血清(Hyclone)+100U/ml青霉素(Sigma)+100μg/ml链霉素(Sigma)+高糖DMDM培养基(Gibco)+forskolin(2μM～25μM)+Repso x(2～15uM)+CHIR99021(1μM～10μM)+VPA(0.5mM～1.5mM)+PDGF-AB(100～250ng/mL)，该培养系统中10％胎牛血清也可以被血清替代品(invitrogen)以10％～20％的浓度替代；100U/ml青霉素(Sigma)和100μg/ml链霉素(Sigma)可以不使用。在37℃，5％CO 2环境下培养细胞。 In the second step, the small molecule compound of the skin fibroblasts is treated as follows: the base culture solution is replaced with a small molecule treatment solution, and the cells are cultured for 2-20 days, and the small molecule treatment solution means: 10% fetal bovine serum (Hyclone) + 100 U/ Ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high sugar DMDM medium (Gibco) + forskolin (2 μM ~ 25 μM) + Repso x (2 ~ 15 uM) + CHIR99021 (1 μM ~ 10 μM) + VPA (0.5 mM ~ 1.5 mM) + PDGF-AB (100 ~ 250 ng / mL), 10% fetal bovine serum in this culture system can also be replaced by serum replacement (invitrogen) at a concentration of 10% to 20%; 100 U / ml penicillin ( Sigma) and 100 μg/ml streptomycin (Sigma) may not be used. The cells were cultured at 37 ° C in a 5% CO 2 atmosphere.
其余步骤同实施例1。The remaining steps are the same as in the first embodiment.
实施例11Example 11
第2步骤中骨髓间充质干细胞的小分子化合物处理为：更换基础培养液为小分子处理液，培养细胞2-20天，小分子处理液是指：10％胎牛血清(Hyclone)+100U/ml青霉素(Sigma)+100μg/ml链霉素(Sigma)+高糖DMDM培养基(Gibco)+forskolin(2μM～25μM)+5-Aza-2'-deoxycytidine(0.5～20μM),该培养系统中10％胎牛血清也可以被血清替代品(invitrogen)以10％～20％的浓度替代；100U/ml青霉素(Sigma)和100μg/ml链霉素(Sigma)可以不使用。在37℃，5％CO 2环境下培养细胞。 In the second step, the small molecule compound of the bone marrow mesenchymal stem cells is treated as follows: the base medium is replaced with a small molecule treatment solution, and the cells are cultured for 2-20 days, and the small molecule treatment solution means: 10% fetal bovine serum (Hyclone) + 100U. /ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high sugar DMDM medium (Gibco) + forskolin (2 μM ~ 25 μM) + 5-Aza-2'-deoxycytidine (0.5 ~ 20 μM), the culture system The 10% fetal bovine serum can also be replaced by a serum replacement (invitrogen) at a concentration of 10% to 20%; 100 U/ml penicillin (Sigma) and 100 μg/ml streptomycin (Sigma) may not be used. Cells were cultured at 37 ℃, 5% CO 2 environment.
其余步骤同实施例2。The remaining steps are the same as in Embodiment 2.
上述实施例中细胞的变化见图1～图11；图1为小分子化合物作用后人源皮肤成纤维细胞的形态变化,A图未处理时,正常培养皮肤成纤维细胞，传到到第13代，一些原来呈长梭形生长的成纤维细胞,开始出现细胞体积变大,细胞形状不规则的老化情况，使用β-半乳糖苷酶染色，B图中可见衰老细胞染色成蓝色；使用如实施例1中的方法处理后，第14代的细胞，形态基本规则地呈长梭形，使用β-半乳糖苷酶染色，结果为阴性。The changes of cells in the above examples are shown in Fig. 1 to Fig. 11; Fig. 1 shows the morphological changes of human skin fibroblasts after the action of small molecule compounds, and when the A picture is not treated, the normal cultured skin fibroblasts are passed to the 13th. Generation, some fibroblasts that originally grew in long spindle shape began to appear with large cell size and irregular cell shape, stained with β-galactosidase, and B showed senescent cells stained blue; After treatment with the method of Example 1, the cells of the 14th generation were substantially spindle-shaped in a regular shape, and stained with β-galactosidase, and the result was negative.
图2为小分子化合物作用后人源骨髓间充质干细胞形态的变化，A图未处理时,正常培养间充质干细胞，传到到第10代，一些原来呈梭形生长的细胞胞体变大，细胞拉长或者不规则型生长，使用β-半乳糖苷酶染色，B图中可见衰老细胞染色成蓝色；使用如实施例2中的方法处理后，第12代的细胞，形态基本规则地呈梭形，使用β-半乳糖苷酶染色，结果为阴性。Figure 2 shows the changes in the morphology of human bone marrow mesenchymal stem cells after the action of small molecule compounds. When the A picture is not treated, the normal cultured mesenchymal stem cells are passed to the 10th generation, and some cell bodies with original spindle-shaped growth become larger. , cell elongated or irregular growth, stained with β-galactosidase, B picture shows senescent cells stained blue; after treatment with the method as in Example 2, the 12th generation of cells, the basic rules The ground was spindle-shaped and stained with β-galactosidase, and the result was negative.
图3为小分子化合物作用后人源皮肤成纤维细胞细胞性状的鉴定，使用实施例1中的方法处理后，细胞和处理前一样，正常表达皮肤成纤维细胞的分子标志物CD29(A图),vimentin(B图)；C图对比了处理前后细胞的增值情况，处理组Fib-treated的增值能力比未处理的Fib要明显。Figure 3 is a diagram showing the cell traits of human dermal fibroblasts after the action of small molecule compounds. After treatment with the method of Example 1, the cells were normally expressed as the molecular marker CD29 of skin fibroblasts (Fig. A). Vimentin (panel B); Figure C compares the value-added of cells before and after treatment. The value-added ability of Fib-treated in the treatment group is more obvious than that of untreated Fib.
图4为小分子化合物作用后人源骨髓间充质干细胞细胞性状的鉴定，如实施例2中，A图为处理前后间充质干细胞的分子标记物的表达，表达阳性的CD29,CD90,CD73以及阴性的CD34,CD45在处理前后的细胞中表达一致；B图中处理后的细胞MSC-treated要比处理前MSC的增值能力好；C图为处理后的MSC细胞，同样正常地进行3系分化，如图中显示了该细胞的成骨和成脂分化。Figure 4 is a diagram showing the cell traits of human bone marrow mesenchymal stem cells after the action of small molecule compounds. As in Example 2, Figure A shows the expression of molecular markers of mesenchymal stem cells before and after treatment, and positive expression of CD29, CD90, CD73. And the negative CD34, CD45 expression in the cells before and after treatment; B-treated cells after treatment MSC-treated better than the pre-treatment MSC value-added; C picture shows the treated MSC cells, also normal 3 series Differentiation, as shown in the figure, shows osteogenesis and adipogenic differentiation of the cells.
图5为通过小分子化合物作用后人源细胞的端粒长度的变化，A图中，以处理前的人源 皮肤成纤维细胞作为对照，采用实施例1、实施例3～10中的方法，端粒长度增加且大于1.5倍；B图中处理了人源骨髓间充质干细胞，采用实施例2和实施例11的方法，处理后端粒长度增加。5 is a change in the telomere length of a human-derived cell by a small molecule compound. In the A-picture, the human skin fibroblasts before treatment are used as a control, and the methods in Example 1 and Examples 3 to 10 are used. The telomere length was increased by more than 1.5 times; human bone marrow mesenchymal stem cells were treated in panel B, and the length of the back end granules was increased by the methods of Example 2 and Example 11.
图6为通过小分子化合物作用后人源细胞的端粒长度和年龄的对比，以及端粒相关基因的表达：与约35岁健康人群的平均端粒长度相比较，以及与自体相比较，52岁个体来源的皮肤成纤维细胞经过小分子组合处理后，其端粒长度增加明显，如A图所示；与端粒延长相关的基因TPP1瞬时高表达，如图B；图C显示，TERT(端粒酶反转录酶)在永生化细胞293T(“1”),Day12的Fib(人源皮肤成纤维细胞；“2”,实施例1)，Day12的MSC(人源骨髓间充质干细胞；“3”，实施例2)中短暂表达。Figure 6 is a comparison of telomere length and age of human cells after treatment with small molecule compounds, and expression of telomere-associated genes: compared with mean telomere length in healthy populations of about 35 years old, and compared with autologous After treatment with small molecules, the telomere length of the individual-derived skin fibroblasts increased significantly, as shown in Figure A; the transiently high expression of the gene TPP1 associated with telomere elongation, as shown in Figure B; Figure C shows that TERT ( Telomerase reverse transcriptase) in immortalized cells 293T ("1"), Day12 Fib (human dermal fibroblasts; "2", Example 1), Day12 MSC (human bone marrow mesenchymal stem cells) "3", transient expression in Example 2).
图7为对小分子化合物作用后人源皮肤成纤维细胞中与衰老相关基因的表达变化，在处理后的细胞中，与衰老相关的基因均下调，例如与P53肿瘤抑制通路相关的基因CDKN1A、ATF3、GADD45B，BTG2，与衰老相关的H2A组蛋白家族成员X；Figure 7 shows the expression changes of aging-related genes in human dermal fibroblasts after treatment with small molecule compounds. In the treated cells, the genes related to aging are down-regulated, for example, the gene CDKN1A associated with the P53 tumor suppressor pathway, ATF3, GADD45B, BTG2, H2A histone family member X associated with aging;
图8为小分子化合物作用后人源皮肤成纤维细胞体外功能活力的检测：如实施例3，皮肤成纤维细胞的I型胶原蛋白，在处理后明显上调；发育早期皮肤细胞的标记物ITGA6,在处理后上调明显。Figure 8 is a graph showing the in vitro functional viability of human dermal fibroblasts after action of a small molecule compound: as in Example 3, type I collagen of skin fibroblasts was significantly up-regulated after treatment; ITGA6, a marker for early developmental skin cells, The up-regulation is obvious after processing.
图9为小分子化合物作用后人源皮肤成纤维细胞体内功能活力的检测：从皮肤分离的成纤维细胞具有较弱的免疫调节能力，通过实施例6的方法处理细胞后，在异体皮肤移植的模型中，将细胞培养48小时后的培养液注射到受体鼠体内，8天后观察皮肤移植部位，与对照组(未处理细胞组Fib，PBS组)相比，供体移植皮肤收到的免疫排斥反应较小，处理后的细胞具有明显的全身性免疫调节作用。Figure 9 is a test for the functional viability of human dermal fibroblasts in vivo after the action of small molecule compounds: fibroblasts isolated from the skin have weak immunomodulatory ability, and after treatment of cells by the method of Example 6, transplantation in allogeneic skin In the model, the culture solution after 48 hours of cell culture was injected into the recipient mice, and the skin graft site was observed 8 days later, and the donor skin was immunized compared with the control group (untreated cell group Fib, PBS group). The rejection is small, and the treated cells have obvious systemic immunomodulatory effects.
图10为对小分子化合物作用后长期传代的细胞干性的检测和成瘤性检测结果图：对实施例1中处理的细胞，进行Nod-SCID小鼠的皮下细胞移植，28天后未见成瘤，如图A白圈内所示；图B中对该组细胞进行干性基因OCT4的染色，未见表达。Fig. 10 is a graph showing the results of detection and tumorigenicity of cell dryness after long-term passage of a small molecule compound: For the cells treated in Example 1, subcutaneous cell transplantation of Nod-SCID mice was performed, and no effect was observed after 28 days. Tumors are shown in the white circle of Figure A; in Figure B, the cells were stained with the dry gene OCT4 and no expression was observed.
图11小分子化合物作用后(如实施例1)人源皮肤成纤维细胞的克隆形成数量高于处理前，如图A所示；图B显示形成的克隆形态，处理前细胞(Fib)的克隆较小，细胞分散；处理过的细胞(Fib-treated)形态紧密，克隆较大。Figure 11 After the action of the small molecule compound (as in Example 1), the number of clones of human skin fibroblasts was higher than that before treatment, as shown in Figure A; Figure B shows the cloned form, clones of pre-treatment cells (Fib) Smaller, cell-dispersed; treated cells (Fib-treated) are tightly packed and larger clones.

Claims (15)

  1. 一种预防、延缓或者逆转哺乳动物如人的细胞、组织、器官或机体衰老进程的小分子化合物组合，其特征在于，所述小分子化合物组合包括DNMT抑制剂、HMT抑制剂、组蛋白去甲基化酶抑制剂、TGF-β抑制剂、WNT/β-catenin激动剂、cAMP激动剂和赖氨酸脱乙酰基酶抑制剂中的至少一种。A combination of small molecule compounds for preventing, delaying or reversing the aging process of cells, tissues, organs or organisms of a mammal such as a human, characterized in that the combination of small molecule compounds comprises DNMT inhibitors, HMT inhibitors, histones At least one of a base enzyme inhibitor, a TGF-β inhibitor, a WNT/β-catenin agonist, a cAMP agonist, and a lysine deacetylase inhibitor.
  2. 如权利要求1所述的小分子化合物组合，其特征在于，所述小分子化合物组合为DNMT抑制剂、HMT抑制剂、组蛋白去甲基化酶抑制剂和赖氨酸脱乙酰基酶抑制剂中的至少一种。The small molecule compound combination according to claim 1, wherein the small molecule compound is a combination of a DNMT inhibitor, an HMT inhibitor, a histone demethylase inhibitor, and a lysine deacetylase inhibitor At least one of them.
  3. 如权利要求1所述的小分子化合物组合，其特征在于，所述小分子化合物组合为TGF-β抑制剂、WNT/β-catenin激动剂、cAMP激动剂、DNMT抑制剂、HMT抑制剂和赖氨酸脱乙酰基酶抑制剂中的至少一种。The small molecule compound combination according to claim 1, wherein the small molecule compound is a TGF-β inhibitor, a WNT/β-catenin agonist, a cAMP agonist, a DNMT inhibitor, an HMT inhibitor, and a La At least one of the amino acid deacetylase inhibitors.
  4. 如权利要求1所述的小分子化合物组合，其特征在于，所述小分子化合物还包括RAR激动剂、ascorbate(抗坏血酸)和ROCK抑制剂中的至少一种。The small molecule compound combination according to claim 1, wherein the small molecule compound further comprises at least one of an RAR agonist, an ascorbate (ascorbic acid), and a ROCK inhibitor.
  5. 如权利要求1所述的小分子化合物组合，其特征在于，所述小分子化合物组合包括按时序分阶段使用的第一阶段化合物和第二阶段化合物，所述第一阶段化合物为DNMT抑制剂、HMT抑制剂、组蛋白去甲基化酶抑制剂、TGF-β抑制剂、WNT/β-catenin激动剂、cAMP激动剂和赖氨酸脱乙酰基酶抑制剂中的至少一种；The small molecule compound combination according to claim 1, wherein the small molecule compound combination comprises a first stage compound and a second stage compound which are used in stages in stages, the first stage compound being a DNMT inhibitor, At least one of an HMT inhibitor, a histone demethylase inhibitor, a TGF-β inhibitor, a WNT/β-catenin agonist, a cAMP agonist, and a lysine deacetylase inhibitor;
    所述第二阶段化合物为DNMT抑制剂、HMT抑制剂、组蛋白去甲基化酶抑制剂、TGF-β抑制剂，WNT/β-catenin激动剂、cAMP激动剂、赖氨酸脱乙酰基酶抑制剂、RAR激动剂、ascorbate(抗坏血酸)和ROCK抑制剂中的至少一种。The second stage compound is a DNMT inhibitor, an HMT inhibitor, a histone demethylase inhibitor, a TGF-β inhibitor, a WNT/β-catenin agonist, a cAMP agonist, a lysine deacetylase At least one of an inhibitor, a RAR agonist, an ascorbate (ascorbic acid), and a ROCK inhibitor.
  6. 如权利要求1所述的小分子化合物组合，其特征在于，所述TGF-β受体抑制剂为I型TGF-β受体抑制剂，所述cAMP激动剂为EPAC/RAP1激动剂。The small molecule compound combination according to claim 1, wherein the TGF-β receptor inhibitor is a type I TGF-β receptor inhibitor, and the cAMP agonist is an EPAC/RAP1 agonist.
  7. 如权利要求书1～6任一所述的小分子化合物组合，其特征在于，所述小分子化合物组合与PDGF-AB组合使用。The small molecule compound combination according to any one of claims 1 to 6, wherein the small molecule compound combination is used in combination with PDGF-AB.
  8. 如权利要求1～7任一所述的小分子化合物组合，其特征在于，所述赖氨酸脱乙酰基酶抑制剂包括sodium phenylbutyrate，butyrate，sodium butyrate，MC1568，CI994(Tacedinaline)，chidamide，CAY10683(SantacruzaMate A)，CUDC-907，M344(Histone Deacetylase Inhibitor III)，LAQ824(NVP-LAQ824，Dacinostat)，Pracinostat(SB939)，VPA(Valproic acid)，Valproic acid sodium salt,Scriptaid，Apicidin，LBH-589(Panobinostat)，MS-275，SAHA(Vorinostat)，Trichostatin(TSA)，Psammaplin A，PCI-24781(Abexinostat)，Rocilinostat(ACY-1215)，Mocetinostat(MGCD0103)，4-Phenylbutyrate(4PB)，splitomicin，SRT1720，resveratrol，Sirtinol，APHA，CI-994，Depudecin，FK-228，HC-Toxin，ITF-2357(Givinostat)，Chidamide，RGFP 966，PHOB，BG45，Nexturastat A，TMP269，CAY10603，MGCD-0103，Niltubacin，PXD-101(Belinostat)，Pyroxamide，Tubacin，EX-527，BATCP，Cambinol，MOCPAC，PTACH，MC1568，NCH51和TC-H106中的至少一种；The small molecule compound combination according to any one of claims 1 to 7, wherein the lysine deacetylase inhibitor comprises sodium phenylbutyrate, butyrate, sodium butyrate, MC1568, CI994 (Tacedinaline), chidamide, CAY10683 (SantacruzaMate A), CUDC-907, M344 (Histone Deacetylase Inhibitor III), LAQ824 (NVP-LAQ824, Dacinostat), Pracinetastat (SB939), VPA (Valproic acid), Valproic acid sodium salt, Scriptaid, Apicidin, LBH-589 ( Panobinostat), MS-275, SAHA (Vorinostat), Trichostatin (TSA), Psammaplin A, PCI-24781 (Abexinostat), Rocilinostat (ACY-1215), Mocetinostat (MGCD0103), 4-Phenylbutyrate (4PB), splitomicin, SRT1720, Resveratrol, Sirtinol, APHA, CI-994, Depudecin, FK-228, HC-Toxin, ITF-2357 (Givinostat), Chidamide, RGFP 966, PHOB, BG45, Nexturastat A, TMP269, CAY10603, MGCD-0103, Niltubacin, PXD At least one of -101 (Belinostat), Pyroxamide, Tubacin, EX-527, BATCP, Cambinol, MOCPAC, PTACH, MC1568, NCH51 and TC-H106;
    所述TGF-β受体抑制剂包括616452，LY2109761，Pirfenidone，Repsox(E-616452)，SB431542，A77-01，Tranilast，Galunisertib(LY2157299)，A8301，GW788388，ITD-1，SD208，SB525334，LY364947，ASP3029，D4476和SB505124中的至少一种；The TGF-beta receptor inhibitors include 616452, LY2109761, Pirfenidone, Repox (E-616452), SB431542, A77-01, Tranilast, Galunisertib (LY2157299), A8301, GW788388, ITD-1, SD208, SB525334, LY364947, At least one of ASP3029, D4476 and SB505124;
    所述WNT/β-catenin激动剂包括MAY-262611，CHIR98014，CHIR99021，LiCl，Li 2CO 3，TD114-2，AZD2858，AZD1080，BIO，Kenpaullone，TWS119，LY2090314，CBM1078，SB216763和AR-A014418中的至少一种； The WNT/β-catenin agonists include MAY-262611, CHIR98014, CHIR99021, LiCl, Li 2 CO 3 , TD114-2, AZD2858, AZD1080, BIO, Kenpaullone, TWS119, LY2090314, CBM1078, SB216763 and AR-A014418 At least one
    所述cAMP激动剂包括EPAC/RAP1激动剂，8-Bromo-cAMP，Dibutyryl-Camp和Sp-8-Br-cAMPs中的至少一种；The cAMP agonist comprises at least one of an EPAC/RAP1 agonist, 8-Bromo-cAMP, Dibutyryl-Camp and Sp-8-Br-cAMPs;
    所述EPAC/RAP1激动剂包括Forskolin，IBMX，Prostaglandin E2(PGE2)，NKH477，8-pCPT-2′-O-Me-cAMP，GSK256066，Apremilast(CC-10004)，Roflumilast，Cilomilast，Rolipram和Milrinone中的至少一种；The EPAC/RAP1 agonists include Forskolin, IBMX, Prostaglandin E2 (PGE2), NKH477, 8-pCPT-2'-O-Me-cAMP, GSK256066, Apremilast (CC-10004), Roflumilast, Cilomilast, Rolipram and Milrinone At least one type;
    所述RAR激动剂包括TTNPB，Bexarotene，Ch55，Tamibarotene，Retinol，AM580，ATRA，13-cis RA，Vitamin A及Vitamin A衍生物中的至少一种；The RAR agonist comprises at least one of TTNPB, Bexarotene, Ch55, Tamibarotene, Retinol, AM580, ATRA, 13-cis RA, Vitamin A and Vitamin A derivatives;
    所述ROCK抑制剂包括Y-27632，Y-27632 2HCl，Thiazovivin，Ripasudil(K-115)，Fasudil，Fasudil(HA-1077)HCl，GSK429286A，RKI-1447和PKI-1313中的至少一种；The ROCK inhibitor comprises at least one of Y-27632, Y-27632 2HCl, Thiazovivin, Ripasudil (K-115), Fasudil, Fasudil (HA-1077) HCl, GSK429286A, RKI-1447 and PKI-1313;
    所述DNMT抑制剂包括RG108，Thioguanine，5-Aza-2'-deoxycytidine(Decitabine)，SGI-1027，Zebularine和5-Azacytidine(AZA)中的至少一种；The DNMT inhibitor comprises at least one of RG108, Thioguanine, 5-Aza-2'-deoxycytidine (Decitabine), SGI-1027, Zebularine and 5-Azacytidine (AZA);
    所述HMT抑制剂包括EPZ004777，EPZ5676，GSK503，BIX 01294，DZNep，DZNep HCL,SGC 0946中的至少一种；The HMT inhibitor comprises at least one of EPZ004777, EPZ5676, GSK503, BIX 01294, DZNep, DZNep HCL, SGC 0946;
    所述组蛋白去甲基化酶抑制剂包括parnate(tranylcypromine)，Tranylcypromine(2-PCPA)HCl SP2509，4SC-202，ORY-1001(RG-6016)，GSKJ1和GSK-LSD1中的至少一种。The histone demethylase inhibitor comprises at least one of parnate (tranylcypromine), Tranylcypromine (2-PCPA) HCl SP2509, 4SC-202, ORY-1001 (RG-6016), GSKJ1 and GSK-LSD1.
  9. 一种包含如权利要求1～8任一所述的小分子化合物组合的试剂盒、培养液/基、药物、保健品、食品、化妆品或医疗器械。A kit, culture solution/base, medicament, health care product, food, cosmetic or medical device comprising the combination of small molecule compounds according to any one of claims 1 to 8.
  10. 一种如权利要求1～8任一所述的小分子化合物组合在预防、延缓或者逆转哺乳动物如人的细胞、组织、器官或机体衰老进程和相关疾病中的应用及提高哺乳动物如人组织、器官损伤修复能力的应用。A combination of small molecule compounds according to any one of claims 1 to 8 for preventing, delaying or reversing the aging process and related diseases of cells, tissues, organs or organisms of mammals such as humans and for improving mammalian tissues such as humans The application of organ damage repair ability.
  11. 如权利要求10所述的应用，其特征在于，所述细胞来源于哺乳动物如人的机体细胞或体外培养的细胞。The use according to claim 10, wherein the cells are derived from a body cell of a mammal such as a human or a cell cultured in vitro.
  12. 一种由权利要求1～8任一所述的小分子化合物组合预防、延缓或者逆转哺乳动物如人的细胞、组织、器官或机体衰老进程的方法。A method for preventing, delaying or reversing the aging process of a cell, tissue, organ or body of a mammal such as a human by the combination of the small molecule compound according to any one of claims 1 to 8.
  13. 一种通过权利要求1～8任一所述的小分子化合物组合作用得到的年轻化的细胞及细胞产物。A younger cell and cell product obtained by the combination of the small molecule compounds according to any one of claims 1 to 8.
  14. 一种如权利要求13所述的年轻化的细胞及细胞产物的应用。A use of the youngened cells and cell products of claim 13.
  15. 一种由权利要求1～8任一所述的小分子化合物组合，其特征在于，所述小分子化合 物组合用于延长哺乳动物如人的细胞的端粒长度，瞬时刺激端粒相关蛋白的表达或上调；作用后不会刺激哺乳动物如人的细胞表达干性基因。A combination of small molecule compounds according to any one of claims 1 to 8, wherein the combination of small molecule compounds is used to prolong telomere length of cells of a mammal such as a human, and transiently stimulate expression of telomere-associated proteins. Or up-regulation; does not stimulate mammalian cells such as humans to express dry genes after action.
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