CN101919877A - 间充质干细胞的新用途 - Google Patents
间充质干细胞的新用途 Download PDFInfo
- Publication number
- CN101919877A CN101919877A CN2010102625767A CN201010262576A CN101919877A CN 101919877 A CN101919877 A CN 101919877A CN 2010102625767 A CN2010102625767 A CN 2010102625767A CN 201010262576 A CN201010262576 A CN 201010262576A CN 101919877 A CN101919877 A CN 101919877A
- Authority
- CN
- China
- Prior art keywords
- stem cells
- mesenchymal stem
- osteoclasts
- bone
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000002901 mesenchymal stem cell Anatomy 0.000 title abstract description 39
- 210000002997 osteoclast Anatomy 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 8
- 210000000130 stem cell Anatomy 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 4
- 208000001132 Osteoporosis Diseases 0.000 claims description 2
- 230000009897 systematic effect Effects 0.000 claims 1
- 238000001727 in vivo Methods 0.000 abstract description 9
- 208000020084 Bone disease Diseases 0.000 abstract description 4
- 230000001105 regulatory effect Effects 0.000 abstract description 3
- 238000000338 in vitro Methods 0.000 abstract description 2
- 230000005764 inhibitory process Effects 0.000 abstract 1
- 210000000988 bone and bone Anatomy 0.000 description 18
- 210000004027 cell Anatomy 0.000 description 18
- 239000002158 endotoxin Substances 0.000 description 18
- 230000006698 induction Effects 0.000 description 18
- 229920006008 lipopolysaccharide Polymers 0.000 description 18
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 16
- 241000699666 Mus <mouse, genus> Species 0.000 description 15
- 241000699670 Mus sp. Species 0.000 description 15
- 239000000243 solution Substances 0.000 description 15
- 238000010186 staining Methods 0.000 description 13
- 239000002609 medium Substances 0.000 description 11
- 239000006143 cell culture medium Substances 0.000 description 10
- 230000006378 damage Effects 0.000 description 10
- 208000027418 Wounds and injury Diseases 0.000 description 9
- 239000008367 deionised water Substances 0.000 description 9
- 229910021641 deionized water Inorganic materials 0.000 description 9
- 230000002757 inflammatory effect Effects 0.000 description 9
- 208000014674 injury Diseases 0.000 description 9
- 239000002953 phosphate buffered saline Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical group O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 238000002347 injection Methods 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 239000012091 fetal bovine serum Substances 0.000 description 7
- 239000012192 staining solution Substances 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- NPGIHFRTRXVWOY-UHFFFAOYSA-N Oil red O Chemical compound Cc1ccc(C)c(c1)N=Nc1cc(C)c(cc1C)N=Nc1c(O)ccc2ccccc12 NPGIHFRTRXVWOY-UHFFFAOYSA-N 0.000 description 5
- 230000004069 differentiation Effects 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- -1 CD31 Proteins 0.000 description 4
- 239000000834 fixative Substances 0.000 description 4
- 238000003306 harvesting Methods 0.000 description 4
- 229940027941 immunoglobulin g Drugs 0.000 description 4
- 230000002188 osteogenic effect Effects 0.000 description 4
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 4
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 229950003937 tolonium Drugs 0.000 description 4
- HNONEKILPDHFOL-UHFFFAOYSA-M tolonium chloride Chemical compound [Cl-].C1=C(C)C(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 HNONEKILPDHFOL-UHFFFAOYSA-M 0.000 description 4
- 210000000689 upper leg Anatomy 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 3
- 241000699679 Cricetulus migratorius Species 0.000 description 3
- 229930182555 Penicillin Natural products 0.000 description 3
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 3
- 102000007591 Tartrate-Resistant Acid Phosphatase Human genes 0.000 description 3
- 108010032050 Tartrate-Resistant Acid Phosphatase Proteins 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- 210000001185 bone marrow Anatomy 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 3
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 3
- 239000000411 inducer Substances 0.000 description 3
- 229940049954 penicillin Drugs 0.000 description 3
- 238000011725 BALB/c mouse Methods 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 102100032912 CD44 antigen Human genes 0.000 description 2
- 102000029816 Collagenase Human genes 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 2
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 2
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 2
- 101001046686 Homo sapiens Integrin alpha-M Proteins 0.000 description 2
- 101000935043 Homo sapiens Integrin beta-1 Proteins 0.000 description 2
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 2
- 102100022338 Integrin alpha-M Human genes 0.000 description 2
- 102100025304 Integrin beta-1 Human genes 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 101001065566 Mus musculus Lymphocyte antigen 6A-2/6E-1 Proteins 0.000 description 2
- 108010004729 Phycoerythrin Proteins 0.000 description 2
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000009815 adipogenic differentiation Effects 0.000 description 2
- 230000002293 adipogenic effect Effects 0.000 description 2
- 210000000845 cartilage Anatomy 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000009816 chondrogenic differentiation Effects 0.000 description 2
- 230000002648 chondrogenic effect Effects 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000003511 endothelial effect Effects 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 230000003394 haemopoietic effect Effects 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 230000009818 osteogenic differentiation Effects 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229910001961 silver nitrate Inorganic materials 0.000 description 2
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 2
- 235000019345 sodium thiosulphate Nutrition 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 210000002303 tibia Anatomy 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- YNXICDMQCQPQEW-UHFFFAOYSA-N 1-naphthyl dihydrogen phosphate Chemical compound C1=CC=C2C(OP(O)(=O)O)=CC=CC2=C1 YNXICDMQCQPQEW-UHFFFAOYSA-N 0.000 description 1
- 208000009304 Acute Kidney Injury Diseases 0.000 description 1
- 101710145634 Antigen 1 Proteins 0.000 description 1
- 102000000503 Collagen Type II Human genes 0.000 description 1
- 108010041390 Collagen Type II Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 206010019663 Hepatic failure Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102100023915 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- 208000033626 Renal failure acute Diseases 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 206010058141 Skin graft rejection Diseases 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- MIJPAVRNWPDMOR-UHFFFAOYSA-N [2-(1,2-dihydroxyethyl)-3-hydroxy-5-oxo-2h-furan-4-yl] dihydrogen phosphate Chemical compound OCC(O)C1OC(=O)C(OP(O)(O)=O)=C1O MIJPAVRNWPDMOR-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 201000011040 acute kidney failure Diseases 0.000 description 1
- 208000012998 acute renal failure Diseases 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- DHCLVCXQIBBOPH-UHFFFAOYSA-N beta-glycerol phosphate Natural products OCC(CO)OP(O)(O)=O DHCLVCXQIBBOPH-UHFFFAOYSA-N 0.000 description 1
- GHRQXJHBXKYCLZ-UHFFFAOYSA-L beta-glycerolphosphate Chemical compound [Na+].[Na+].CC(CO)OOP([O-])([O-])=O GHRQXJHBXKYCLZ-UHFFFAOYSA-L 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 210000001612 chondrocyte Anatomy 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 1
- 229960003957 dexamethasone Drugs 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 230000011132 hemopoiesis Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000013394 immunophenotyping Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 208000007903 liver failure Diseases 0.000 description 1
- 231100000835 liver failure Toxicity 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- DDRCIGNRLHTTIW-UHFFFAOYSA-N n-(4-amino-2,5-dimethoxyphenyl)benzamide Chemical compound C1=C(N)C(OC)=CC(NC(=O)C=2C=CC=CC=2)=C1OC DDRCIGNRLHTTIW-UHFFFAOYSA-N 0.000 description 1
- 230000024121 nodulation Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000003938 response to stress Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Substances [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 1
- 229960001471 sodium selenite Drugs 0.000 description 1
- 235000015921 sodium selenite Nutrition 0.000 description 1
- 239000011781 sodium selenite Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了间充质干细胞的新用途。该新用途是离体的间充质干细胞在制备具有抑制破骨细胞生成功能的产品中的应用。本发明拓展了间充质干细胞的用途,建立了一种利用间充质干细胞调节体内破骨细胞活动的方法,在骨疾病预防和治疗领域将有广阔的应用前景。
Description
技术领域
本发明涉及间充质干细胞的新用途。
背景技术
间充质干细胞是一类最早在骨髓中被发现的干细胞,随后在全身多处组织中被分离鉴定出来。间充质干细胞在体外可以大量扩增,在体内可以分化为多种组织细胞。另外,间充质干细胞还能够支持造血和抑制免疫反应。基于以上优点,它已经成为组织工程、细胞治疗和干细胞技术等方面的重要种子细胞,具有广阔的应用前景。目前,间充质干细胞已经被用于治疗糖尿病,急性肾衰竭,脓毒血症,心肌梗塞,皮肤移植排异和肝衰竭等疾病。
破骨细胞是一类起源于造血干细胞的成体细胞,它们大部分贴附于骨表面,可以分泌多种酸和溶解酶来降解骨组织。破骨细胞的作用具有双面性:在机体代谢稳定时,只有少量的破骨细胞生成,进而与成骨细胞一起,参加骨组织的新陈代谢。当机体处于疾病状态时,大量的破骨细胞生成,参与机体应激反应,炎性损伤以及肿瘤骨转移等病理过程。因此,寻找调节破骨细胞的发育异常和功能紊乱的有效方法,是一项治疗相关疾病的重要策略。
脂多糖炎性损伤小鼠是一种体内破骨细胞生成的动物模型。给予小鼠输注脂多糖后,其体内各种免疫细胞(如淋巴细胞和巨噬细胞等)发生活化,相应炎性细胞因子如TNF-α,IL-1β分泌增加,破骨细胞的生成显著活跃,骨质破坏作用增强。
发明内容
本发明的一个目的是提供间充质干细胞的一种新用途。
本发明所提供的一种新用途是离体的间充质干细胞在制备具有抑制破骨细胞生成功能的产品中的应用。
上述应用中,所述间充质干细胞为骨实质来源的间充质干细胞。
上述应用中,所述产品为药物。
本发明所提供的另一种新用途是离体的间充质干细胞在制备具有治疗和/或预防骨疾病功能的产品中的应用。
上述应用中,所述间充质干细胞为骨实质来源的间充质干细胞。
上述应用中,所述骨疾病为骨质疏松症。
上述应用中,所述产品为药物。
上述任一所述的间充质干细胞可从商业途径得到,也可以自己分离制备,可为骨髓来源的,也可以为骨实质来源的。
本发明发明人基于对间充质干细胞特性的认识,选择了脂多糖炎性损伤小鼠来研究间充质干细胞对于体内破骨细胞生成的调节作用。结果表明间充质干细胞可以抑制炎性微环境中破骨细胞的生长。本发明拓展了间充质干细胞的用途,建立了一种利用间充质干细胞调节体内破骨细胞活动的方法,在骨疾病预防和治疗领域将有广阔的应用前景。
附图说明
图1为细胞免疫表型分析。
图2为成骨分化鉴定结果。
图3为成脂分化鉴定结果。
图4为成软骨分化鉴定结果。
图5为各组切片染色结果。
图6为分别注射了磷酸盐缓冲液、脂多糖、脂多糖联合间充质干细胞的小鼠体内破骨细胞数量的比较。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
本发明所有动物实验严格尊重军事医学科学实验动物指南进行。
实施例1、小鼠间充质干细胞的分离培养
C57BL/6雌性小鼠购自军事医学科学院实验动物中心。二型胶原酶购自Sigma公司。α-MEM培养基从Hyclone公司购得。谷氨酰胺从Sigma公司购得。
取2周龄健康C57BL/6雌性小鼠,脱颈处死,75%酒精浸泡5分钟后,无菌条件下分离小鼠股骨和胫骨,去净表面组织,以注射器吸取含10%体积胎牛血清(从Hyclone公司购得)的磷酸盐缓冲液(氯化钠8g/L,磷酸氢二钠2.9g/L,氯化钾0.2g/L,磷酸二氢钾0.24g/L,溶剂为去离子水)冲净骨髓腔,小心剪碎为大小为2mm3细小骨片,置细胞培养基I中[细胞培养基I由二型胶原酶、胎牛血清和α-MEM培养基组成,二型胶原酶在细胞培养基I中的浓度为0.15%(质量百分比),胎牛血清在细胞培养基I中的浓度为20%(体积百分比)],在37℃、5%CO2条件下消化1小时后种骨片于细胞培养基II中[细胞培养基II由谷氨酰胺、胎牛血清和α-MEM培养基组成,谷氨酰胺在细胞培养基II中的浓度为2mmol/L,胎牛血清在细胞培养基中的浓度为10%(体积百分比)],在为37℃、5%CO2条件下培养72小时后,首次换液(用细胞培养基II替换旧液),去除悬浮细胞,保留骨片,从骨片中生长出的细胞长满80%培养瓶面积后消化传代(用细胞培养基II传代培养),连续传代,取第4-6代细胞做体内注射用。
间充质干细胞的鉴定:按照文献方法(Zhu H,Guo ZK,Jiang XX,Li H,Wang XY,Yao HY,Zhang Y,Mao N.A protocol for isolation and culture of mesenchymal stem cellsfrom mouse compact bone.Nature Protocols.2010;5(3):550-560.)进行所得小鼠间充质干细胞的鉴定。即:
(一)、MSCs的细胞免疫表型分析
1、准备细胞:以胰酶消化收获第四代细胞,制备成单细胞悬液,调整细胞数量至1×106细胞/EP管,洗净后重悬至100μLPBS/EP管。
2、抗体标记:按照说明书要求,如表1.1所示,分别加入FITC标记的抗小鼠CD11b,CD45和Sca-1,PE标记的抗小鼠CD29,CD31,CD34,CD44,CD86,CD105,Ia和上述抗体相对应的同型抗体。4℃避光孵育30分钟。同时避光染PI,15分钟。
表1.1 MSCs免疫表型分析相关抗体及应用
对应缩写:Armenian Hamster IgG=Armenian Hamster immunoglobulin G,亚美尼亚仓鼠免疫球蛋白G;CD=cluster of differentiation,分化群;FITC=fluorescein isothiocyanate,异硫氰酸荧光素;PE=phycoerythrin,藻红素;Rat IgG=rat immunoglobulin G,大鼠免疫球蛋白G;Sca-1=stem cell antigen-1,干细胞抗原1;
3、流式分析:用PBS,4℃,300G,8分钟,洗细胞两遍后,上机分析。
结果如图1所示。PI染色,用于流式分析的MSCs活细胞比率大于95%。利用流式细胞术分析小鼠骨实质来源MSCs的免疫学表型,发现其具有经典的MSCs表型特点。高表达干细胞标记Sca-1和CD105;间质标记CD29和CD44,低表达或者不表达造血标记CD11b,CD34和CD45;内皮标记CD31;共刺激分子CD86和Ia。从表型特点上判断,小鼠骨实质来源的MSCs是一群非造血非内皮间质来源的干细胞。
(二)、MSCs的多向诱导分化及鉴定
1、成骨分化
1)成骨诱导:收获第四代MSCs,按照1×104/孔种于24孔培养板内,对照组培养体系为α-MEM、10%FBS、100U/mL青霉素、100U/mL链霉素,诱导组培养体系为对照组体系添加表1.2中诱导剂。每2-3天换液。
表1.2 成骨诱导体系
2)碱性磷酸酶染色:诱导第14天,去除培养基;向培养板内加入固定液(柠檬酸40μL+去离子水2mL+丙酮3mL)固定30秒;向培养板内加入染色液(坚牢兰RR 62.5μL+去离子水3mL+As-Mx 125μL);避光反应30分钟;倒掉染液,加少量PBS,显微镜下照相。
3)Von Kossa染色:诱导第28天,去除培养基;加入4%多聚甲醛固定30分钟;洗去固定液,加入5%硝酸银溶液,强光下反应30分钟;洗净后,加入5%硫代硫酸钠溶液,反应5分钟;洗净后,显微镜下拍照。
结果:(图2a)成骨诱导14天,MSCs中的碱性磷酸酶活性显著上调,染色后成红色;(图2b)继续成骨诱导到28天,有明显的骨结节形成(图中微标尺代表100μm)。
2、成脂分化
1)成脂诱导:收获第四代MSCs,按照2×104/孔种于24孔培养板内,对照组培养体系为α-MEM、10%FBS、100U/mL青霉素、100U/mL链霉素,诱导组培养体系为对照组体系添加表1.3中诱导剂。每2-3天换液。
表1.3 成脂诱导体系
2)油红O染色:诱导第14天,去除培养基;向培养板内加入染色液(油红O粉剂0.5g+异丙醇100mL);避光反应15分钟;倒掉染液,加少量PBS,显微镜下照相。
结果:油红O具有亲脂肪特性,(图3)诱导的第14天进行油红O染色,可见着色的脂滴充满了胞质(图中微标尺代表100μm)。
3、成软骨分化
1)成软骨诱导:收获第四代MSCs,按照1×106/管种于15mL塑料离心管内,对照组培养体系为HG-DMEM、10%FBS、100U/mL青霉素、100U/mL链霉素,诱导组培养体系为对照组体系添加表1.4中诱导剂。在4℃,以300G离心6分钟。每2-3天换液。
表1.4 成软骨诱导体系
2)甲苯胺蓝染色:诱导第21天,去除培养基;取出软骨小球,10%福尔马林固定1小时;
常规蜡块包埋,做5μM病理切片;切片脱蜡,复水;向切片上加入染色液(甲苯胺蓝粉剂0.5g+去离子水100mL);反应15分钟;倒掉染液,去离子水洗净,显微镜下照相。
结果:(图4)对软骨小球进行切片染色,可以看到丰富的细胞外基质呈环形包裹着细胞小球,甲苯胺蓝染色后阳性,基质中软骨细胞形态良好(图中微标尺代表200μm)。
综合以上结果,证明分离得到的细胞就是骨实质来源的间充质干细胞。
MSC鉴定所需试剂和设备如下:
1、MSCs免疫表型鉴定试剂
抗体均为eBioscience公司产品:
-FITC-labeled anti-stem cell antigen-1(Sca-1)
-FITC-labeled anti-CD 11b
-PE-labeled anti-CD29
-PE-labeled anti-CD31
-PE-labeled anti-CD34
-PE-labeled anti-CD44
-FITC-labeled anti-CD45
-PE-labeled anti-CD86
-PE-labeled anti-CD105
-PE-labeled anti-Ia
-FITC-labeled rat IgG2b isotype control
-PE-labeled rat IgG2b isotype control
-PE-labeled Armenian Hamster IgG isotype control
-FITC-labeled rat IgG2a isotype control
-PE-labeled rat IgG2a isotype control
碘化丙啶(Propidium iodide,PI)为Sigma公司产品。
2、MSCs诱导分化及鉴定试剂
1)MSCs诱导分化试剂
高糖-DMEM(HG-DMEM)培养基为Hyclone公司产品,地塞米松、维生素C磷酸盐、β-磷酸甘油、IBMX、胰岛素、转铁蛋白、亚硒酸钠和丙酮酸盐为Sigma公司产品,TGF-β3为R&D公司产品。
2)鉴定试剂:油红O染液、碱性磷酸酶检测试剂盒、硝酸银、硫代硫酸钠和甲苯胺蓝为Sigma公司产品。
3、仪器设备:25cm2/75cm2培养瓶(corning-costar),24孔细胞培养板(Falcon,costar),15mL塑料离心管(corning),细胞培养箱(Thermo Forma),流式细胞仪(CoulterEPICS XL,BD),生物净化工作台(北京中化生物技术研究所/伟达净化技术研究所,X90-3),倒置显微镜(Olympus),DNA thermal cycler(Perkin-Elmer),台式离心机(Beckman),低温高速离心机(GS-1 5R Beckman),分光光度计(BIO-RAD),解剖剪,解剖镊,无菌纱布,0.4mm1mL注射器,1.5mL EP管,35mm和100mm玻璃平皿。
实施例2、构建体内破骨细胞生成的模型
脂多糖购自Sigma公司,产品目录号为L2630;健康BALB/c小鼠购自军事医学科学院实验动物中心;
取6-8周健康BALB/c小鼠,把50μg脂多糖溶解于200μL磷酸盐缓冲液内,得到脂多糖溶液;将脂多糖溶液经腹腔注射到小鼠体内,正常饲养1小时后,得到炎性损伤小鼠;取实施例1中获得的第4-6代间充质干细胞,经尾静脉输入炎性损伤小鼠体内,正常饲养5天后,检测小鼠体内破骨细胞的生成数量。
试验分A,B,C三组:
A组(PBS):单只小鼠注射200μL磷酸盐缓冲液,不注射间充质干细胞;
B组(LPS):单只小鼠注射脂多糖溶液(注射剂量:200μL/只),不注射间充质干细胞;
C组(LPS+MSCs):单只小鼠注射脂多糖溶液(注射剂量:200μL/只),1个小时后,注射间充质干细胞(注射剂量:1×106个间充质干细胞/只)。
检测小鼠体内破骨细胞的生成数量的方法如下:
(1)小鼠股骨切片的制备
本发明所有动物实验严格遵守军事医学科学实验动物指南进行。间充质干细胞注射5天后,将小鼠脱颈处死,75%酒精浸泡5分钟后,无菌条件下取出股骨和胫骨,以脱钙液10mL固定2天,石蜡包埋,切5μm厚的组织切片,脱蜡,复水。
(2)抗酒石酸的酸性磷酸酶染色
用抗酒石酸的酸性磷酸酶测定试剂盒(Sigma-Aldrich产品)对各组切片进行染色,光学显微镜下观察破骨细胞形态拍照,对破骨细胞进行计数和统计学分析(T测验)。检测细胞数量的方法:1)、把切片放入固定液中作用30秒,(固定液=3mL丙酮+1.8mL去离子水+0.2mL柠檬酸);2)、取出切片,加入去离子水洗三遍,空气中晾干;3)、把切片放入染色液中,避光,37℃,染一小时(染色液=3.9mL去离子水+0.2mL酒石酸+0.2mL萘酚磷酸盐溶液+0.2mL醋酸溶液+0.5ml染色胶囊溶液);4)、取出切片,加入去离子水洗三遍;5)、切片中深酒红色的细胞判定为抗酒石酸酸性磷酸酶染色阳性,为破骨细胞。在显微镜下对贴附于骨小梁及骨内膜表面的破骨细胞进行拍照,计数。
各组的切片染色结果如图5所示(图中微标尺代表100μm。)。结果:采用间充质干细胞能有效地抑制脂多糖炎性损伤小鼠体内的破骨细胞生成。
破骨细胞数量统计学分析结果如图6所示(*P<0.05,具有统计学意义)。结果:与没有给予间充质干细胞的脂多糖小鼠炎性损伤相比较,输入间充质干细胞的脂多糖炎性损伤小鼠股骨切片上的破骨细胞显著减少,说明间充质干细胞显著的抑制了体内破骨细胞的生成。
PBS本身是一种不具有生物活性的缓冲液,在本发明中作为脂多糖的溶剂。正常小鼠体内应该具有生理水平的少量的破骨细胞,接受脂多糖后,破骨细胞数量大量增加。设立PBS组实际是更为严格的对照,比采用正常小鼠作为对照更为严格,排除了注射损伤本身等因素对体内破骨细胞活动的影响。
Claims (7)
1.离体的间充质干细胞在制备具有抑制破骨细胞生成功能的产品中的应用。
2.根据权利要求1所述的应用,其特征在于:所述间充质干细胞为骨实质来源的间充质干细胞。
3.根据权利要求1或2所述的应用,其特征在于:所述产品为药物。
4.离体的间充质干细胞在制备具有治疗和/或预防骨疾病功能的产品中的应用。
5.根据权利要求4所述的应用,其特征在于:所述间充质干细胞为骨实质来源的间充质干细胞。
6.根据权利要求4或5所述的应用,其特征在于:所述骨疾病为骨质疏松症。
7.根据权利要求4-6中任一所述的应用,其特征在于:所述产品为药物。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2010102625767A CN101919877A (zh) | 2010-07-30 | 2010-08-24 | 间充质干细胞的新用途 |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201010241971.7 | 2010-07-30 | ||
CN201010241971 | 2010-07-30 | ||
CN2010102625767A CN101919877A (zh) | 2010-07-30 | 2010-08-24 | 间充质干细胞的新用途 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN101919877A true CN101919877A (zh) | 2010-12-22 |
Family
ID=43335261
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN2010102625767A Pending CN101919877A (zh) | 2010-07-30 | 2010-08-24 | 间充质干细胞的新用途 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN101919877A (zh) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101486992A (zh) * | 2008-12-05 | 2009-07-22 | 中国人民解放军军事医学科学院基础医学研究所 | 抑制破骨细胞生成的间充质干细胞及其制备方法与应用 |
-
2010
- 2010-08-24 CN CN2010102625767A patent/CN101919877A/zh active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101486992A (zh) * | 2008-12-05 | 2009-07-22 | 中国人民解放军军事医学科学院基础医学研究所 | 抑制破骨细胞生成的间充质干细胞及其制备方法与应用 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108671224B (zh) | 血小板裂解物的组合物、用途和制备 | |
Crisan et al. | A perivascular origin for mesenchymal stem cells in multiple human organs | |
US12053492B2 (en) | Perinatal tissue derived mesenchymal stem cells: method of preparation and uses thereof | |
JP6348848B2 (ja) | 間葉系幹細胞の増殖 | |
US20220110979A1 (en) | Fibroblast regenerative cells | |
EP3385368B1 (en) | Method for producing mesenchymal stem cells | |
TW200902718A (en) | Procurement, isolation, and cryopreservation of endometrial/menstrual cells | |
WO2017094260A1 (ja) | 脊椎動物の脂肪組織由来間葉系細胞株の製造方法 | |
JP6193214B2 (ja) | 歯髄由来の多能性幹細胞の製造方法 | |
He et al. | Comparative study of mesenchymal stem cells from rat bone marrow and adipose tissue | |
AU2013206755B2 (en) | Activating adipose-derived stem cells for transplantation | |
JP2022524764A (ja) | 免疫調節性間葉系幹細胞 | |
US9650604B2 (en) | Equine amniotic membrane-derived mesenchymal stem cells | |
CN103710305B (zh) | 使用含有海藻糖的细胞洗涤溶液的贴壁细胞的洗涤方法 | |
CN101919877A (zh) | 间充质干细胞的新用途 | |
JP2021523242A (ja) | 細胞の高張液に由来する細胞外物質に関連する方法及び組成物 | |
RU2795621C2 (ru) | Способ получения клеток из зубной пульпы | |
Van Phuc et al. | Improving the Efficacy of Diabetes Mellitus Treatment by Combining Cell Replacement Therapy with Immune Correction | |
Midolo | Immunoregulatory properties of bone marrow mesenchymal stromal cell-derived extracellular vesicles | |
Akhlaq | Journey of mesenchymal stem cells in biomedical research: Current aspects and scenario | |
HK1195744B (zh) | 血小板裂解物的組合物、用途和製備 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C12 | Rejection of a patent application after its publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20101222 |