CN113106057B - 一种用于肝细胞移植的自体干细胞制剂及制备方法 - Google Patents
一种用于肝细胞移植的自体干细胞制剂及制备方法 Download PDFInfo
- Publication number
- CN113106057B CN113106057B CN202011537970.7A CN202011537970A CN113106057B CN 113106057 B CN113106057 B CN 113106057B CN 202011537970 A CN202011537970 A CN 202011537970A CN 113106057 B CN113106057 B CN 113106057B
- Authority
- CN
- China
- Prior art keywords
- urine
- cell
- cells
- liver
- stem 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.)
- Active
Links
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 73
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 238000002054 transplantation Methods 0.000 title abstract description 26
- 210000005229 liver cell Anatomy 0.000 title abstract description 18
- 210000004027 cell Anatomy 0.000 claims abstract description 127
- 210000002700 urine Anatomy 0.000 claims abstract description 66
- 239000001963 growth medium Substances 0.000 claims abstract description 21
- 230000004069 differentiation Effects 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 6
- 210000004185 liver Anatomy 0.000 claims description 34
- 230000006698 induction Effects 0.000 claims description 19
- 239000006143 cell culture medium Substances 0.000 claims description 18
- 239000006285 cell suspension Substances 0.000 claims description 13
- 210000003494 hepatocyte Anatomy 0.000 claims description 13
- 238000005406 washing Methods 0.000 claims description 13
- 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 claims description 12
- 239000002609 medium Substances 0.000 claims description 11
- 239000000243 solution Substances 0.000 claims description 11
- 230000002440 hepatic effect Effects 0.000 claims description 10
- 210000002966 serum Anatomy 0.000 claims description 7
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 6
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 6
- 229960000723 ampicillin Drugs 0.000 claims description 6
- 238000007664 blowing Methods 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 claims description 6
- 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 claims description 6
- 229960005322 streptomycin Drugs 0.000 claims description 6
- 239000012091 fetal bovine serum Substances 0.000 claims description 4
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 4
- 239000002504 physiological saline solution Substances 0.000 claims description 4
- 229930024421 Adenine Natural products 0.000 claims description 3
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 3
- 241000283690 Bos taurus Species 0.000 claims description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- 102000009024 Epidermal Growth Factor Human genes 0.000 claims description 3
- 101800003838 Epidermal growth factor Proteins 0.000 claims description 3
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 claims description 3
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims description 3
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 claims description 3
- 102000004877 Insulin Human genes 0.000 claims description 3
- 108090001061 Insulin Proteins 0.000 claims description 3
- 108010019160 Pancreatin Proteins 0.000 claims description 3
- AUYYCJSJGJYCDS-LBPRGKRZSA-N Thyrolar Chemical compound IC1=CC(C[C@H](N)C(O)=O)=CC(I)=C1OC1=CC=C(O)C(I)=C1 AUYYCJSJGJYCDS-LBPRGKRZSA-N 0.000 claims description 3
- 102000004338 Transferrin Human genes 0.000 claims description 3
- 108090000901 Transferrin Proteins 0.000 claims description 3
- 229960000643 adenine Drugs 0.000 claims description 3
- 238000004113 cell culture Methods 0.000 claims description 3
- 229960003957 dexamethasone Drugs 0.000 claims description 3
- 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 claims description 3
- 229940116977 epidermal growth factor Drugs 0.000 claims description 3
- 229960000890 hydrocortisone Drugs 0.000 claims description 3
- 229940125396 insulin Drugs 0.000 claims description 3
- 210000002510 keratinocyte Anatomy 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000010899 nucleation Methods 0.000 claims description 3
- 229940055695 pancreatin Drugs 0.000 claims description 3
- 230000001817 pituitary effect Effects 0.000 claims description 3
- 239000004017 serum-free culture medium Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 230000001502 supplementing effect Effects 0.000 claims description 3
- 239000012581 transferrin Substances 0.000 claims description 3
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 claims description 3
- 238000010370 cell cloning Methods 0.000 claims description 2
- 102100021866 Hepatocyte growth factor Human genes 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 239000013049 sediment Substances 0.000 claims 1
- 208000019423 liver disease Diseases 0.000 abstract description 33
- 210000002901 mesenchymal stem cell Anatomy 0.000 abstract description 15
- 210000005228 liver tissue Anatomy 0.000 abstract description 11
- 210000001185 bone marrow Anatomy 0.000 abstract description 9
- 230000035755 proliferation Effects 0.000 abstract description 9
- 238000005516 engineering process Methods 0.000 abstract description 7
- 230000008901 benefit Effects 0.000 abstract description 5
- 230000004071 biological effect Effects 0.000 abstract description 4
- 206010052779 Transplant rejections Diseases 0.000 abstract description 3
- 238000001727 in vivo Methods 0.000 abstract description 3
- 230000003908 liver function Effects 0.000 abstract description 3
- 230000007774 longterm Effects 0.000 abstract description 3
- 238000012136 culture method Methods 0.000 abstract description 2
- 230000002980 postoperative effect Effects 0.000 abstract description 2
- 208000037816 tissue injury Diseases 0.000 abstract description 2
- 208000027418 Wounds and injury Diseases 0.000 abstract 1
- 238000010186 staining Methods 0.000 description 21
- 208000010334 End Stage Liver Disease Diseases 0.000 description 11
- 208000011444 chronic liver failure Diseases 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 9
- 206010019663 Hepatic failure Diseases 0.000 description 8
- 229930040373 Paraformaldehyde Natural products 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 230000006870 function Effects 0.000 description 8
- 208000007903 liver failure Diseases 0.000 description 8
- 231100000835 liver failure Toxicity 0.000 description 8
- 229920002866 paraformaldehyde Polymers 0.000 description 8
- 208000007788 Acute Liver Failure Diseases 0.000 description 6
- 206010000804 Acute hepatic failure Diseases 0.000 description 6
- 241000699660 Mus musculus Species 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 238000000465 moulding Methods 0.000 description 6
- 238000011580 nude mouse model Methods 0.000 description 6
- 230000012292 cell migration Effects 0.000 description 5
- 238000012258 culturing Methods 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 238000011476 stem cell transplantation Methods 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 231100000836 acute liver failure Toxicity 0.000 description 4
- 230000007547 defect Effects 0.000 description 4
- 230000007850 degeneration Effects 0.000 description 4
- 239000000975 dye Substances 0.000 description 4
- 230000035876 healing Effects 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 230000009818 osteogenic differentiation Effects 0.000 description 4
- 230000007170 pathology Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 210000003462 vein Anatomy 0.000 description 4
- 208000007342 Diabetic Nephropathies Diseases 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 206010067125 Liver injury Diseases 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 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 3
- 241000209094 Oryza Species 0.000 description 3
- 235000007164 Oryza sativa Nutrition 0.000 description 3
- RGCKGOZRHPZPFP-UHFFFAOYSA-N alizarin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 description 3
- 235000013339 cereals Nutrition 0.000 description 3
- 230000002648 chondrogenic effect Effects 0.000 description 3
- 208000019425 cirrhosis of liver Diseases 0.000 description 3
- 210000000805 cytoplasm Anatomy 0.000 description 3
- 208000033679 diabetic kidney disease Diseases 0.000 description 3
- 239000000835 fiber Substances 0.000 description 3
- MOFVSTNWEDAEEK-UHFFFAOYSA-M indocyanine green Chemical compound [Na+].[O-]S(=O)(=O)CCCCN1C2=CC=C3C=CC=CC3=C2C(C)(C)C1=CC=CC=CC=CC1=[N+](CCCCS([O-])(=O)=O)C2=CC=C(C=CC=C3)C3=C2C1(C)C MOFVSTNWEDAEEK-UHFFFAOYSA-M 0.000 description 3
- 229960004657 indocyanine green Drugs 0.000 description 3
- 235000009566 rice Nutrition 0.000 description 3
- 239000012192 staining solution Substances 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- 102100022464 5'-nucleotidase Human genes 0.000 description 2
- 102100023126 Cell surface glycoprotein MUC18 Human genes 0.000 description 2
- 102000009016 Cholera Toxin Human genes 0.000 description 2
- 108010049048 Cholera Toxin Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 102100037241 Endoglin Human genes 0.000 description 2
- 206010018367 Glomerulonephritis chronic Diseases 0.000 description 2
- 229920002527 Glycogen Polymers 0.000 description 2
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 2
- 102000003745 Hepatocyte Growth Factor Human genes 0.000 description 2
- 101000678236 Homo sapiens 5'-nucleotidase Proteins 0.000 description 2
- 101000623903 Homo sapiens Cell surface glycoprotein MUC18 Proteins 0.000 description 2
- 101000881679 Homo sapiens Endoglin Proteins 0.000 description 2
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 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
- 101000884271 Homo sapiens Signal transducer CD24 Proteins 0.000 description 2
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 description 2
- 102100025304 Integrin beta-1 Human genes 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 2
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 2
- 102100038081 Signal transducer CD24 Human genes 0.000 description 2
- 108010017842 Telomerase Proteins 0.000 description 2
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 description 2
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 230000009815 adipogenic differentiation Effects 0.000 description 2
- 230000011759 adipose tissue development Effects 0.000 description 2
- 208000010002 alcoholic liver cirrhosis Diseases 0.000 description 2
- 230000000735 allogeneic effect Effects 0.000 description 2
- 239000001045 blue dye Substances 0.000 description 2
- 230000007910 cell fusion Effects 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 210000002889 endothelial cell Anatomy 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 229940096919 glycogen Drugs 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 2
- 208000006359 hepatoblastoma Diseases 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 230000003907 kidney function Effects 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 230000007102 metabolic function Effects 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 230000002188 osteogenic effect Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 208000009304 Acute Kidney Injury Diseases 0.000 description 1
- 208000022309 Alcoholic Liver disease Diseases 0.000 description 1
- 206010003827 Autoimmune hepatitis Diseases 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 208000000419 Chronic Hepatitis B Diseases 0.000 description 1
- 206010057573 Chronic hepatic failure Diseases 0.000 description 1
- 206010009208 Cirrhosis alcoholic Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 208000010228 Erectile Dysfunction Diseases 0.000 description 1
- 102100031806 Fas-binding factor 1 Human genes 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 102000037280 Growth Differentiation Factor 2 Human genes 0.000 description 1
- 108010090290 Growth Differentiation Factor 2 Proteins 0.000 description 1
- 206010019799 Hepatitis viral Diseases 0.000 description 1
- 208000002972 Hepatolenticular Degeneration Diseases 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000012654 Primary biliary cholangitis Diseases 0.000 description 1
- 208000033626 Renal failure acute Diseases 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 241000168254 Siro Species 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 201000011040 acute kidney failure Diseases 0.000 description 1
- 231100000439 acute liver injury Toxicity 0.000 description 1
- 208000012998 acute renal failure Diseases 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000002293 adipogenic effect Effects 0.000 description 1
- 210000004504 adult stem cell Anatomy 0.000 description 1
- 210000004381 amniotic fluid Anatomy 0.000 description 1
- 239000002870 angiogenesis inducing agent Substances 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000003445 biliary tract Anatomy 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000022159 cartilage development Effects 0.000 description 1
- 210000005056 cell body Anatomy 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 239000002458 cell surface marker Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 208000006990 cholangiocarcinoma Diseases 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 231100000012 chronic liver injury Toxicity 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
- 230000003021 clonogenic effect Effects 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 230000005757 colony formation Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000003235 crystal violet staining Methods 0.000 description 1
- JVHIPYJQMFNCEK-UHFFFAOYSA-N cytochalasin Natural products N1C(=O)C2(C(C=CC(C)CC(C)CC=C3)OC(C)=O)C3C(O)C(=C)C(C)C2C1CC1=CC=CC=C1 JVHIPYJQMFNCEK-UHFFFAOYSA-N 0.000 description 1
- ZMAODHOXRBLOQO-UHFFFAOYSA-N cytochalasin-A Natural products N1C(=O)C23OC(=O)C=CC(=O)CCCC(C)CC=CC3C(O)C(=C)C(C)C2C1CC1=CC=CC=C1 ZMAODHOXRBLOQO-UHFFFAOYSA-N 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 230000009986 erectile function Effects 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 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 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 231100000234 hepatic damage Toxicity 0.000 description 1
- 208000007386 hepatic encephalopathy Diseases 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000004957 immunoregulator effect Effects 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 201000001881 impotence Diseases 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000008818 liver damage Effects 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 235000003170 nutritional factors Nutrition 0.000 description 1
- 230000011164 ossification Effects 0.000 description 1
- 210000004738 parenchymal cell Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000001023 pro-angiogenic effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000001044 red dye Substances 0.000 description 1
- 210000005084 renal tissue Anatomy 0.000 description 1
- 238000006748 scratching Methods 0.000 description 1
- 230000002393 scratching effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 210000005070 sphincter Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 210000003954 umbilical cord Anatomy 0.000 description 1
- 210000002229 urogenital system Anatomy 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 201000001862 viral hepatitis Diseases 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
- C12N2500/24—Iron; Fe chelators; Transferrin
- C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/40—Nucleotides, nucleosides or bases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/12—Hepatocyte growth factor [HGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/39—Steroid hormones
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/395—Thyroid hormones
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Developmental Biology & Embryology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Genetics & Genomics (AREA)
- Animal Behavior & Ethology (AREA)
- Wood Science & Technology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Immunology (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Epidemiology (AREA)
- Rheumatology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供了一种用于肝细胞移植的自体干细胞制剂，从慢性肝病患者自身尿液中提取出尿源性干细胞，其生物学性状不受肝脏疾病本身的影响，具有强大的增殖能力，多次传代扩增后仍具有干细胞特性和多向分化潜能，体内移植可显著改善肝脏功能、修复肝组织损伤，作为自体的干细胞，不存在免疫排斥，且获取无创，作为肝细胞移植治疗的种子细胞较骨髓间充质干细胞有较大优势。本发明还提供了一种上述自体干细胞制剂的制备方法，分离培养方法简单方便，患者3‑4天的24小时尿液中获取的尿源性干细胞，传至P4‑P6代可获得足够细胞数量用于移植。本发明还提供了一种上述尿源性干细胞的培养基，配方经过优化后，价格便宜，配制方便，获得的细胞克隆数更多。该方法能有效获得大量自体干细胞，细胞可随时并多次获取，减少了干细胞长期贮存的费用，有望解决目前肝细胞移植技术中细胞来源缺乏及术后移植排斥反应的难题，具有重大的社会经济效益。
Description
技术领域：
本发明属于组织工程领域，具体涉及一种用于肝细胞移植的自体干细胞制剂。
技术背景：
各种慢性病毒性肝炎、自身免疫性肝炎、代谢性肝病、酒精性肝病、肝癌等所致的慢性肝损害进展的最终结局都是肝功能衰竭。肝功能衰竭是临床最常见的死亡率极高的肝病症候群，严重威胁着人类的健康。据统计显示，我国每年因为重症肝功能衰竭导致死亡的人数为30-50万。肝移植是目前唯一有效治愈肝衰竭的方法，但由于供体缺乏，手术并发症、排斥反应风险较大，而且费用昂贵，无法满足众多肝衰竭患者的治疗需求。肝细胞移植是将体外分离纯化的具有正常生物活性的肝实质细胞，通过各种不同途径导入受体，使之发挥正常生理代谢功能的一种细胞工程技术。大量的动物实验及初步临床研究证明，肝细胞移植是治疗肝功能衰竭，肝性脑病，肝细胞功能缺陷的遗传代谢性疾病的一种潜在治疗方法。卫生部将干细胞技术归入“第三类医疗技术”，认为其“涉及重大伦理问题，安全性、有效性尚需经规范的临床试验研究进一步验证”。然而，目前人成熟肝细胞或胎肝细胞来源的困难、异种哺乳动物肝细胞免疫源性高和传播动物源性传染病的可能性、永生化肝细胞株的功能差异以及远期致瘤危险等，使细胞来源成为肝细胞移植技术进一步用于临床研究与应用的瓶颈。
干细胞已被证实具有强大的分化潜能、自我更新能力、免疫调节作用以及靶向治疗功能，现有研究表明，其可以在肝衰竭治疗中发挥重要作用。依据细胞的来源不同，目前研究治疗肝衰竭的干细胞主要有以下几种：肝成体干细胞也被称为肝卵圆细胞或肝祖细胞前体细胞，间充质干细胞(包括骨髓间充质干细胞、脂肪间充质干细胞、脐带间充质干细胞、脐血间充质干细胞、羊水间充质干细胞等)，胚胎干细胞等。
目前用于肝脏疾病研究的干细胞来源存在伦理上的争议或者有创性方面的不足之处，而且通常来自健康人的捐助，虽然同种异体间充质干细胞的抗原性相对较小，但是患者仍然可能会对来自异体的细胞产生免疫排斥反应以及发生移植相关的副作用，甚至加速病情进展。理想的种子细胞应使用来源于患者自身的干细胞，可不被自身免疫系统所排斥。由于慢性肝衰竭患者多为中老年，脐带造血干细胞储存时间为20年，难以满足大多数慢性肝衰竭患者的自体干细胞移植的需求。目前国内外已有大量应用自体骨髓间充质干细胞移植治疗慢性肝衰竭的临床试验病例，然而疗效报道不一，仍需进一步的研究以证实其在终末期肝病中的作用。并且骨髓间充质干细胞采集需要患者的整体状态良好，并提前进行细胞动员，MSCs的获取必须使用侵入性的有创方法，这会增加患者不必要的痛苦和潜在并发症的风险。如果能找到一种更易获取的自体干细胞，作为肝细胞移植的种子细胞，将会在很大程度上避免以上不足。
尿液中存在一类为数不多却具有很强增殖功能以及具有某些间充质干细胞特征的细胞(Yuanyuan Zhang et al.Urine derived cells are a potential source forurological tissue reconstruction.J Urol.2008；180(5):2226-2233)，称之为尿源性干细胞(Urine derived cells,USCs)。与骨髓间充质干细胞相比，USCs更易获取，无创，费用低；USCs具有较高端粒酶活性，其自我更新和细胞增殖能力比骨髓间充质干细胞强一倍，能保持约达68次群体倍增，收集24h尿液即可获得140个USC克隆，理论上来说，在培养4周后，可产生细胞数量(5.3×1010，P5-P6代)，足够临床应用；经数代培养后，染色体数目仍保持正常，未见肿瘤细胞的形成；USCs具有更强地分泌营养因子的功能，如分泌促血管生长因子和细胞因子；USCs具有免疫调节作用，可抑制外周血中T细胞，B细胞的增殖。作为一种新型的功能完全的干细胞。目前，USCs已应用于泌尿生殖系统(如膀胱缺损，急性肾衰竭，糖尿病肾病，阴茎勃起功能障碍等)的组织工程研究，在不同的模型中能改善肾功能，阴茎勃起功能，以及尿道括约肌功能等。USCs在肝脏的微环境中有向肝细胞分化的潜能，体外诱导USCs成肝分化，ALB-Gluc(白蛋白启动的荧光素酶报告基因)活性明显增强，部分细胞具有吲哚菁绿摄取和糖原合成功能，USCs移植入肝脏可在一定程度上改善损伤肝脏的功能及修复肝脏组织。
USCs是来源于肾脏的干细胞，在肾脏疾病累及肾脏组织结构时，难以获得正常USCs。糖尿病肾病及慢性肾炎患者USCs的生物学性状，与同年龄的正常健康人相比，患者尿液中获取的USCs克隆数减少，细胞形态杂乱，细胞倍增时间延长，端粒酶活性下降，分泌的细胞因子量减少(陶立，等.糖尿病肾病患者尿源性干细胞的分离鉴定及与健康人尿源性干细胞的细胞生物学比较研究.中国细胞生物学学报.2018，40(2)：194-201)。慢性肝病患者的肾脏组织结构和肾功能正常，理论上来说不会影响USCs的增殖分化等生物学活性。但有研究发现慢性肝衰竭患者自体骨髓间充质细胞的体外增殖能力明显低于非慢性肝病患者。
研究内容：
为了解决的现有技术中存在的细胞来源困难、数量不足，获取有创、体内移植排斥反应等缺陷，本发明提供了一种用于肝细胞移植的自体干细胞制剂及其制备方法。
本发明的目的是通过以下措施实现的：
一种用于肝细胞移植的自体干细胞制剂，包含尿源性干细胞和缓冲液。
上述尿源性干细胞是从慢性肝病患者尿液中提取、分离、培养获得。
进一步地，上述慢性肝病包括：肝炎后肝硬化、酒精性肝硬化、胆汁淤积性肝硬化、慢性肝功能衰竭、多囊肝、先天性胆道闭塞、肝豆状核变性、肝母细胞瘤、肝细胞肝癌、胆管细胞癌、肝血管内皮癌。
上述尿源性肝细胞呈米粒状和/或纺锤状生长，细胞群表达CD24、CD29、CD73、CD90、CD146，不表达或低表达CD31、CD34、CD45、CD105。
上述缓冲液为含有1-3％患者自身血清的生理盐水或PBS。
上述自体干细胞制剂中的细胞浓度为1-2×107个细胞/毫升。
本发明还提供了一种上述自体干细胞制剂的制备方法，包括以下步骤：
步骤1：收集200mL无菌清洁中段尿液，加入40mL尿源性干细胞培养基，分装至50mL离心管，1500rpm，离心5min；
步骤2：弃上清，保留细胞沉淀，每个离心管加入2-4mL尿源性干细胞培养基，吹打成细胞悬液，补充尿源性干细胞培养基至24mL，轻轻吹匀后种入24孔细胞培养板，1mL/孔，置于5％CO2的细胞培养箱，37℃培养；
步骤3：10-14天，出现细胞克隆，将24孔板中所有细胞传代至60mm培养板，此后每3-5天，待细胞生长至90％以上，按1:3-4比例传代；
步骤4：分次收集无菌清洁中段尿液共2000-4000mL，重复步骤1至3，选取P4-P6代尿源性干细胞，用0.25％胰酶/EDTA消化后，PBS或生理盐水洗涤3次，缓冲液重悬制成含有1-2×107个细胞/毫升的细胞悬液。
可选地，将P4-P6代尿源性干细胞进行成肝诱导分化后制成细胞悬液，具体地，在含有2％马血清，0.1nmol/mL地塞米松，10ng/mL肝细胞生长因子，20ng/mL碱性成纤维细胞生长因子4，100U/mL氨苄青霉素及1mg/mL链霉素的DMEM培养基中诱导10-14天。优化地，尿源性干细胞在成肝诱导培养基中与正常肝祖细胞或正常成熟肝细胞分离共培养。
本发明还提供了一种尿源性干细胞培养基，由角质形成细胞无血清培养基、DMEM培养基、Hamm’s F12培养基及胎牛血清按体积比500：337：113：50组成，含牛垂体提取物20μg/mL、2.5μg/mL胰岛素、2.5μg/mL转铁蛋白、0.24μg/mL氢化可的松、136ng/mL 3,3′,5-三碘-L-甲状腺原氨酸、5ng/mL表皮生长因子、1μmol/mL谷氨酰胺、90nmol/mL腺嘌呤、100U/mL氨苄青霉素及1mg/mL链霉素。
有益效果：
1.本发明提供了一种用于肝细胞移植的自体干细胞制剂，包含从慢性肝病患者自身尿液中提取出的USCs，其生物学性状不受肝脏疾病本身的影响，具有强大的增殖能力，多次传代扩增后仍具有干细胞特性和多向分化潜能，体内移植可显著改善肝脏功能、修复肝组织损伤，作为自体的干细胞，不存在免疫排斥，且获取无创，作为肝细胞移植治疗的种子细胞较骨髓间充质干细胞有较大优势。
2.本发明提供了一种上述自体干细胞制剂的制备方法，USCs分离培养方法简单方便，24小时尿液中获取的细胞在P4-P6代可获得足够细胞数量用于移植。尿源性干细胞的培养基配方经过优化后，价格便宜，配制方便，与现有方案培养基比较，获得的细胞克隆数更多。该方法能有效获得大量自体干细胞，细胞可随时并多次获取，减少了干细胞长期贮存的费用，有望解决目前肝细胞移植技术中细胞来源缺乏及术后移植排斥反应的难题，具有重大的社会经济效益。
说明书附图：
图1正常健康人USCs与慢性肝病患者USCs的细胞形态(白色箭头：米粒状，黑色箭头：纺锤状)
图2正常健康人USCs与慢性肝病患者USCs不同代数细胞的群体倍增时间和每72小时群体倍增次数
图3正常健康人USCs与慢性肝病患者USCs的细胞克隆形成能力
图4正常健康人USCs与慢性肝病患者USCs的细胞划痕愈合能力(A)及transwell迁移能力(B)
图5正常健康人USCs与慢性肝病患者USCs的成骨、成软骨、成脂分化能力
图6正常健康人USCs与慢性肝病患者USCs的成肝分化能力
图7正常健康人USCs与慢性肝病患者USCs移植急性肝衰竭模型的H.E染色
图8正常健康人USCs与慢性肝病患者USCs移植慢性肝衰竭模型的H.E染色(A)及Masson染色(B)
图9Hoechst标记的USCs尾静脉移植后14天定位在肝脏组织中
图10现有方案及本专利的尿源性干细胞培养基培养USCs的增殖(A)、克隆形成(B)、transwell小室迁移(C)及多向分化能力(D)
具体实施方式：
下面通过实施例对本发明进行具体描述，有必要在此指出的是，以下实施例只用于对本发明进行进一步的说明，不能理解为对本发明保护范围的限制，该领域的技术熟练人员可以根据上述发明内容对本发明作出一些非本质的改进和调整。
1.尿源性干细胞的分离、培养
配置尿源性干细胞培养基：按角质形成细胞无血清培养基、DMEM培养基、Hamm’sF12培养基及胎牛血清按体积比500：337：113：50配置，加入牛垂体提取物20μg/mL、2.5μg/mL胰岛素、2.5μg/mL转铁蛋白、0.24μg/mL氢化可的松、136ng/mL 3,3′,5-三碘-L-甲状腺原氨酸、5ng/mL表皮生长因子、1μmol/mL谷氨酰胺、90nmol/mL腺嘌呤、100U/mL氨苄青霉素及1mg/mL链霉素。
收集200mL无菌清洁中段尿液，加入40mL尿源性干细胞培养基，分装至多个50mL离心管，1500rpm，离心5min。弃上清，保留细胞沉淀，每个离心管加入2-4mL尿源性干细胞培养基，吹打成细胞悬液，将多管细胞悬液移至一个50mL离心管，补充尿源性干细胞培养基至24mL，轻轻吹匀后种入24孔细胞培养板，1mL/孔，置于5％CO2的细胞培养箱，37℃培养。10-14天，出现细胞克隆，每200mL尿液中平均获得18-22个细胞克隆，将24孔板中所有细胞传代至6mm培养板，此后每3-5天，待细胞生长至90％以上，按1:3-4比例传代。传至P3代可获得约0.98-2.97×107个细胞，传至P5代可获得0.91-2.67×108个细胞，每24小时尿液(1500-2500mL)收集的细胞克隆传至P5可获得约0.72-3.34×109个细胞。收集患者3-4天的24h尿液，传至P4-P6即可获得足够数量的细胞，满足干细胞移植的要求(1-2×108个/Kg)。选取P4-P6代尿源性干细胞，用0.25％胰酶/EDTA消化后，PBS或生理盐水洗涤3次，缓冲液重悬制成含有1-2×107个细胞/毫升的细胞悬液。
2.慢性肝病患者尿源性干细胞的鉴定及生物学性状检测
从20名慢性肝病患者(年龄55.6±14.5岁，包括：慢性乙肝合并肝硬化，酒精性肝硬化，原发性肝癌，肝母细胞瘤，黄疸性肝炎)的尿液中获取USCs，与相同年龄段(50.5±12.7岁)获取的20名正常健康人的USCs比较，均采用P4-P6代细胞。
流式细胞术检测细胞表面标志：将USCs制成细胞悬液(1×106/管)，PBS洗两遍后每管加入5μL抗体(鼠抗人CD24-FITC、CD29-PE、CD31-FITC、CD34-PE、CD45-FITC、CD73-PE、CD90-PE、CD105-FIT、CD146-PE)，空白对照为不添加抗体的细胞悬液，阴性对照为FITCMouse IgG1、k Isotype Ctrl(FC)和PE Mouse IgG1、k Isotype Ctrl(FC)，室温避光孵育30min，PBS洗涤2次后加300μL PBS重悬，流式细胞仪上机检测。
检测细胞增殖能力：将细胞接种于6孔板，初始浓度为1×105细胞/孔，每组随机选取10个USCs，设置复孔。记录每一代细胞初始数、终末数及每一代相对应的培养时间(精确到半小时)。细胞群体倍增量(population doubling，PD)和细胞群体倍增时间(doublingtime，DT)，通过以下公式进行计算：PD＝ln(Nf/Ni)/ln(2)，DT＝Ct/PD(Nf，细胞终末数；Ni，细胞起始数；Ct，培养时间)。台盼蓝据染实验计数活细胞：将细胞制成细胞悬液，加入10％浓度为0.4％的台盼蓝染液混匀，取20μL至血球计数板，于10×物镜显微镜下观察计数板四角大方格中的细胞数，只计数活细胞(活细胞为透明，死细胞为蓝染)，计算活细胞总数：活细胞数/毫升原液＝(4大格细胞数之和/4)×104，每孔重复计数3次以上。
克隆形成实验：细胞消化后重悬，计数活细胞，于12孔板内每孔接种活细胞400个，每组3个复孔。培养7-10d，计数大于50个细胞的克隆数，用4％多聚甲醛固定后结晶紫染色拍照，计算克隆形成率＝细胞克隆数/接种细胞数×100％。
检测细胞迁移能力：划痕实验：细胞接种于6孔板中，接种密度约为60％，培养18-24h细胞融合率达100％时，用枪头进行划痕，PBS洗3次去除漂浮细胞，加入新鲜培养基，分别于0、12、24h在同一部位观察划痕愈合情况，显微镜下拍照。测量不同时间划痕宽度，计算划痕愈合率(％)＝(0h划痕宽度-实验时间划痕宽度)/0h划痕宽度×100％，实验重复3次。Transwell迁移实验：细胞消化后用无血清培养基重悬，血球计数板计数后稀释细胞悬液至每100μL含约5×104个细胞，吹匀后分别取100μL悬液加入24孔板中的transwell上室，同时下室加入500μL含5％FBS的培养基，每组三个复孔，放入37℃培养箱中培养48h后吸弃上室及下室孔内培养基，PBS洗3次后下室每孔加4％多聚甲醛500μL，室温固定30min，PBS洗3次后下室每孔加500μL结晶紫染液，室温孵育30min，取出小室，用湿棉签轻拭去上室细胞，PBS洗净，显微镜下拍照。
检测细胞多向分化能力：细胞接种于24孔板，接种密度20-30％，分别采用成骨，成脂和成软骨培养基(赛业公司HUXMA-90021，90041，90031)诱导培养。
诱导7天后，ALP染色检测早期成骨分化：吸弃培养基，PBS洗3次，4％多聚甲醛固定30min，PBS洗3次，加入配制好的ALP染色工作液，在37℃孵箱孵育30min，吸弃染液，PBS洗3次，加1mL PBS，显微镜下拍照。
诱导14天后，茜素红染色检测成骨分化：吸弃原培养基，PBS洗3次，4％多聚甲醛固定30min，PBS洗3次，加入1％的茜素红染液，在37℃孵箱孵育30min，吸弃染液，PBS洗3次，加1mL PBS，显微镜下拍照。
诱导14d后进行阿利新蓝染色检测成软骨分化：吸弃原培养基，PBS洗3次，4％多聚甲醛固定30min，PBS洗3次，加入阿利新蓝染液，在37℃孵箱孵育30min，吸弃染液，PBS洗3次，加1mL PBS，显微镜下拍照。
诱导21d后进行油红O染色检测成脂分化:吸弃原培养基，PBS洗3次，4％多聚甲醛固定30min，PBS洗3次，加入油红O染液，在37℃孵箱孵育30min，吸弃染液，PBS洗3次，加1mLPBS，显微镜下拍照。
检测细胞的成肝分化能力：细胞接种于6孔板，接种密度20-30％，采用成肝诱导培养基(含有2％(V/V)马血清、0.1nmol/mL地塞米松、20ng/mL碱性成纤维细胞生长因子4、10ng/mL肝细胞生长因子、5ng/mL骨形态发生蛋白9、100U/mL氨苄青霉素及1mg/mL链霉素的DMEM培养基)诱导培养10-14天，提取RNA，检测ALB、CK18、TAT的表达。细胞接种于24孔板，接种密度20-30％，采用成肝诱导培养基诱导10-14天后，吲哚菁绿摄取释放实验检测细胞代谢功能：DMSO溶解吲哚菁绿粉末制成50mg/mL的ICG储存液，实验前用USCs培养基稀释为1mg/mL的工作浓度，吸弃原培养基，PBS洗2次，每孔加入的ICG工作液200μL，37℃孵育1h，吸弃工作液，PBS洗2次，显微镜下拍照；吸弃PBS，加入USCs培养基放回37℃培养箱，6h后显微镜观察ICG的释放情况。PAS染色检测糖原合成功能：吸弃原培养基，PBS洗2次；每孔加入200μL4％多聚甲醛固定10min，吸弃多聚甲醛，ddH2O轻轻冲洗2min，加入200μL高碘酸液，孵育5min，ddH2O轻轻冲洗2min，加入200μL Schiff染液，孵育15min，ddH2O轻轻冲洗干净；加PBS，显微镜下拍照。可选择地，USCs在24孔板中与肝祖细胞分离共培养，肝祖细胞来源于小鼠胚胎肝脏(Bi Y et al.Functional characteristics of reversibly immortalizedhepatic progenitor cells derived from mouse embryonic liver.Cell PhysiolBiochem.2014；34(4):1318-1338.)，放于transwell上室，USCs放于下室，诱导培养基同上培养12天。
患者每200mL尿液中获取的细胞克隆数为23.96±12.3(n＝20)，正常健康人每200mL尿液中获取的21.2±5.2(n＝20)，细胞克隆数个体差异较大，且与尿液在体外放置时间及温度有关，但每个人的尿液中均可获得细胞克隆。
USCs为贴壁生长细胞，呈米粒状或纺锤状，胞体较小，排列紧密，原代接种后3天左右开始贴壁，10-14天可达50％-60％，即可传代。传代后细胞加速生长，3-4d细胞融合率达80-90％，米粒状细胞比例逐渐减少，纺锤状细胞比例增多，体外培养至10代生长速度明显减慢，细胞扁平失去传代能力(图1)，，正常健康人的USCs与慢性肝病患者的USCs，各组随机选取10例进行传代及细胞形态检测，发现两组USCs的细胞形态无差异。
USCs表达间充质干细胞表面标志物CD24、CD29、CD73、CD90，表达内皮细胞表面标志物CD146，几乎不表达或低表达造血细胞表面标志物CD31、CD34、CD45及内皮细胞标志物CD105，正常健康人USCs与慢性肝病患者USCs的表面标志物表达水平无差异(表1)。
表1.正常健康人USCs与慢性肝病患者USCs的表面标志物表达水平(n＝5)
连续10代培养发现，细胞群体倍增量在正常健康人USCs组(25.65±2.33)与慢性肝病患者USCs组(26.93±1.96)间无统计学差异(P>0.05)，细胞群体倍增时间在两组各代细胞间无统计学差异(图2)。正常健康人USCs组的克隆形成率(9.92±2.27％)，与慢性肝病患者USCs组(9.13±2.88％)组间无统计学差异(图3)。正常健康人USCs组的划痕愈合率(72.2±4.5％)与慢性肝病患者USCs组(69.8±2.7％)间无统计学差异，transwell细胞迁移在两组间无统计学差异(图4)。提示慢性肝病不影响患者尿源性干细胞的表面标志物表达，细胞增殖、克隆形成及细胞迁移能力。
分化实验结果显示：USCs诱导7d后ALP染色阳性率为30-40％，胞质成蓝紫色，核外围较深染，说明有早期成骨分化；诱导14d后茜素红染色阳性，钙结节呈红色，部分细胞为紫红色，说明USCs晚期成骨分化明显，有钙盐沉积。成软骨诱导14天后，两组中几乎所有细胞阿利新蓝染色均呈阳性，胞质及胞膜均呈蓝色。成脂诱导14d后，油红O染色阳性，25-30％的细胞呈空泡样、脂肪样变，红染，部分细胞可见较多脂滴。USCs具有成骨、成软骨及成脂的多向分化潜能，两组间USCs的分化能力无明显差异(图5)。
未诱导的USCs的ICG及PAS染色阴性，经成肝诱导培养基诱导10d后，诱导组有约20％的细胞摄取ICG，表现为胞核绿染，约30％细胞PAS染色阳性，胞浆内出现紫红色颗粒。与肝祖细胞共培养的USCs诱导组细胞ICG阳性率30％以上，PAS染色阳性率约40％。说明USCs具有向一定功能的肝细胞分化的能力，与肝祖细胞共培养对USCs的成肝分化有促进作用，两组间USCs的成肝分化能力无明显差异(图6)。
3.慢性肝病患者的尿源性干细胞移植治疗急性和慢性肝衰竭小鼠模型
急性肝衰竭模型：裸鼠共48只，正常组6只，其余42只给予10％CCL4按20mL/kg剂量腹腔注射1次造模。造模后1天及3天经尾静脉给予正常健康人USCs或慢性肝病患者USCs移植，共两次，造模后1天、3天及7天处死裸鼠，检测肝脏指数，血清ALT及AST，肝脏病理H.E染色。
急性肝衰竭小鼠肝脏指数较正常组明显增高，血清ALT及AST显著增高(表2，p<0.05)，HE染色结果显示对照组裸鼠肝细胞排列规则，结构正常；模型组肝细胞水泡变性，体积增大，气球样变，胞质疏松透亮呈网状，甚至胞核消失。造模后3天，USCs移植组一次移植，模型组肝脏指数及ALT/AST下降(p<0.05)，肝细胞仍有水泡变性，USCs移植对ALT/AST有一定改善，对肝脏指数及肝组织病理的作用不明显，造模后7天，USCs移植组两次移植，模型组肝脏指数基本恢复正常，ALT/AST进一步下降，肝脏病理显著恢复，肝细胞轻度肿胀；USCs移植组肝脏指数基本恢复正常，与模型组有统计学差异，ALT/AST恢复，与模型组无统计学差异，肝细胞排列规则，无明显异常。慢性肝病患者USCs移植组的肝脏指数，ALT/AST及病理改变与正常健康人USCs组无统计学差异(表2，图7)。
表2急性肝衰竭模型各组肝脏指数，ALT及AST水平
a:与对照组相比，p<0.05；b:与同一时间点的模型组相比，p<0.05(n＝6)
慢性肝衰竭小鼠模型：裸鼠共24只，正常组6只，其余18只给予10％CCL4按10mL/kg剂量腹腔注射每周2次造模，8周16次注射后，尾静脉给予正常健康人USCs或慢性肝病患者USCs(2×106个)移植，每周2次，共四次，第一次细胞移植后14天处死裸鼠，检测肝脏指数，血清ALT及AST，肝脏病理H.E染色，Masson染色。
模型组肝脏指数较正常组明显增高(p<0.05)，急性肝损伤标志ALT有增高，但无统计学意义(p>0.05)，慢性肝损伤标志AST增高，有统计学意义(p<0.05)。
USCs移植组与模型组区别：肝脏指数及ALT降低，但无统计学意义(p>0.05)，AST降低，有统计学意义(p<0.05)。H.E染色结果显示对照组裸鼠肝脏组织结构正常，肝小叶完整；模型组有明显的肝细胞退行性变，表现为肝索结构紊乱，核固缩或消失，有较多岛屿状假小叶结构，汇管区有炎性细胞侵润。Masson染色切片可以看到CCL4造模组较空白对照组有明显的纤维增生，汇管区周边纤维架桥形成表现为蓝染，肝脏组织结构紊乱。USCs移植组肝脏组织结构部分恢复正常，肝组织中蓝染纤维显著减少，正常健康人USCs与慢性肝病患者USCs移植组间，肝脏指数，ALT及AST无统计学差异(表3，图8)。
表3慢性肝衰竭模型各组肝脏指数，ALT及AST水平
a:与对照组相比，p<0.05；b:与模型组相比，p<0.05a(n＝6)
以上结果提示：尾静脉移植的USCs能够部分到达肝组织(图9)，对急性或慢性肝衰竭小鼠的肝脏指数及肝脏功能有一定修复作用，特别是对肝组织细胞结构和肝纤维化有显著的改善，慢性肝病患者USCs的移植修复能力与正常健康人USCs相比无显著差异。
4.尿源性干细胞培养基培养细胞
由于参考文献(Yuanyuan Zhang et al.Urine derived cells are a potentialsource for urological tissue reconstruction.J Urol.2008,180(5):2226-2233.)中的尿源性干细胞培养基配置方案中的霍乱毒素属于危险化学品，具有一定毒性，不易购买且存在操作风险，本技术方案优化了尿源性干细胞培养基，去除霍乱毒素，添加了谷氨酰胺，并改良了其他组分的配比。
取10例200mL尿液样本，每例尿液样本平均分成两份，分别采用现有方案及本专利的尿源性干细胞培养基培养。本方案获得的细胞克隆数(10.0±1.13个/100mL)较现有方案获得的细胞克隆数(8.5±0.78/100mL)多，但无统计学差异，在两种不同培养基培养下细胞的增殖、克隆形成、transwell小室迁移及多向分化能力无显著差异(图10)。提示优化后的尿源性干细胞培养基能代替现有方案进行尿源性干细胞的培养。
Claims (4)
1.一种尿源性干细胞培养基，其特征在于：由角质形成细胞无血清培养基、DMEM培养基、Hamm’s F12培养基及胎牛血清按体积比500：337：113：50 组成，含20μg/mL牛垂体提取物、2.5μg/mL 胰岛素、2.5μg/mL 转铁蛋白、0.24μg/mL氢化可的松、136ng/mL 3, 3′, 5-三碘-L-甲状腺原氨酸、5ng/mL表皮生长因子、1 μmol/mL谷氨酰胺、90nmol/mL腺嘌呤、100U/mL氨苄青霉素及1 mg/mL 链霉素。
2.一种自体干细胞制剂的制备方法，其特征在于所述自体干细胞制剂包含尿源性干细胞及缓冲液，制备尿源性干细胞包括以下步骤：
步骤1，收集200mL无菌清洁中段尿液，加入40mL如权利要求1所述的尿源性干细胞培养基，分装至50mL离心管，1500rpm，离心5min；
步骤2，弃上清，保留细胞沉淀，每个离心管加入2-4 mL尿源性干细胞培养基，吹打成细胞悬液，补充尿源性干细胞培养基至24mL，轻轻吹匀后种入24孔细胞培养板，1mL/孔，置于5% CO2的细胞培养箱，37℃培养；
步骤3，10-14天，出现细胞克隆，将24孔板中所有细胞传代至60mm培养板，此后每3-5天，待细胞生长至90%以上，按1: 3-4比例传代；
步骤4，分次收集无菌清洁中段尿液共2000-4000mL，重复步骤1至3，选取P4-P6代尿源性干细胞，用0.25%胰酶/EDTA消化后，PBS或生理盐水洗涤3次，缓冲液重悬制成含有1-2×107个细胞/毫升的细胞悬液。
3.根据权利要求2所述的制备方法，其特征在于还包括以下步骤：将P4-P6代尿源性干细胞进行成肝诱导分化后制成细胞悬液，在含有2%马血清，0.1nmol/mL地塞米松，10ng/mL肝细胞生长因子，20ng/mL 碱性成纤维细胞生长因子4，100U/mL 氨苄青霉素及1mg/mL 链霉素的DMEM培养基中诱导10-14天。
4.根据权利要求3所述的制备方法，其特征在于：尿源性干细胞在成肝诱导培养基中与正常肝祖细胞或正常成熟肝细胞分离共培养。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011537970.7A CN113106057B (zh) | 2020-12-23 | 2020-12-23 | 一种用于肝细胞移植的自体干细胞制剂及制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011537970.7A CN113106057B (zh) | 2020-12-23 | 2020-12-23 | 一种用于肝细胞移植的自体干细胞制剂及制备方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113106057A CN113106057A (zh) | 2021-07-13 |
| CN113106057B true CN113106057B (zh) | 2023-04-25 |
Family
ID=76709498
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011537970.7A Active CN113106057B (zh) | 2020-12-23 | 2020-12-23 | 一种用于肝细胞移植的自体干细胞制剂及制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113106057B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118262798B (zh) * | 2024-05-31 | 2024-08-16 | 山东翰康医学科技集团有限公司 | 一种干细胞质量评价系统 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101052299A (zh) * | 2004-01-27 | 2007-10-10 | 利莱恩斯生命科学有限公司 | 来自角膜缘的未分化干细胞组织系统 |
| CN107254432A (zh) * | 2017-08-16 | 2017-10-17 | 四川大学华西医院 | 一种同时分离尿源性干细胞两种亚群的培养基、分离方法及应用 |
| CN114438026A (zh) * | 2022-02-28 | 2022-05-06 | 广州华越肾科再生医学科技有限公司 | 一种尿源性干细胞的培养基、培养方法及其应用 |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ITFI20060099A1 (it) * | 2006-04-28 | 2007-10-29 | Azienda Ospedaliera Careggi | Popolazione di cellule staminali renali, sua identificazione ed uso per finalita' terapeutiche |
| RU2510276C1 (ru) * | 2012-09-25 | 2014-03-27 | Московский Государственный Университет им. М.В. Ломоносова, Биологический факультет | Способ получения клеток для заместительной клеточной терапии патологий печени |
| CN109576216A (zh) * | 2017-09-29 | 2019-04-05 | 四川大学华西医院 | 尿来源间充质干细胞的提取及扩增培养方法 |
| CN107723271B (zh) * | 2017-10-20 | 2018-09-21 | 杭州观梓健康科技有限公司 | 一种促进诱导人多能干细胞分化为肝样细胞的方法及应用 |
| CN108096186A (zh) * | 2017-12-27 | 2018-06-01 | 重庆斯德姆生物技术有限公司 | 一种肝干细胞注射液及其制备方法 |
| CN110499279B (zh) * | 2019-01-25 | 2021-06-04 | 北京中医药大学 | 一种诱导人尿源性干细胞向肝细胞分化的方法 |
-
2020
- 2020-12-23 CN CN202011537970.7A patent/CN113106057B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101052299A (zh) * | 2004-01-27 | 2007-10-10 | 利莱恩斯生命科学有限公司 | 来自角膜缘的未分化干细胞组织系统 |
| CN107254432A (zh) * | 2017-08-16 | 2017-10-17 | 四川大学华西医院 | 一种同时分离尿源性干细胞两种亚群的培养基、分离方法及应用 |
| CN114438026A (zh) * | 2022-02-28 | 2022-05-06 | 广州华越肾科再生医学科技有限公司 | 一种尿源性干细胞的培养基、培养方法及其应用 |
Non-Patent Citations (2)
| Title |
|---|
| HongFei Xiang等.Exosomes Derived from Human Urine-Derived Stem Cells Inhibit Intervertebral Disc Degeneration by Ameliorating Endoplasmic Reticulum Stress.Oxidative Medicine and Cellular Longevity.2020,摘要. * |
| 苏炜良等.人髓核细胞诱导人尿源性干细胞向髓核样细胞分化作用的研究.中国脊柱脊髓杂志.2020,30(11),1027-1036页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113106057A (zh) | 2021-07-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11135248B2 (en) | Stem cells from urine and methods for using the same | |
| Pereira et al. | Reproducible methodology for the isolation of mesenchymal stem cells from human umbilical cord and its potential for cardiomyocyte generation | |
| Wang et al. | Cell-to-cell contact induces mesenchymal stem cell to differentiate into cardiomyocyte and smooth muscle cell | |
| CN103805562B (zh) | 培养胎盘间充质干细胞的无血清培养基 | |
| Qihao et al. | Spheroid formation and differentiation into hepatocyte-like cells of rat mesenchymal stem cell induced by co-culture with liver cells | |
| US20190264179A1 (en) | Serum-free medium inducing differentiation of umbilical cord mesenchymal stem cell into insulin-secretion-like cell and preparation method and use thereof | |
| US20100330047A1 (en) | Mesenchymal Stem Cells Grown Under Hypoxic Conditions: Compositions, Methods and Uses Therefor | |
| CN106754674A (zh) | 从人胎盘羊膜制备羊膜间充质干细胞的方法及其应用 | |
| WO2012068710A1 (zh) | 从微量人脂肪组织提取间充质干细胞及规模化培养的方法 | |
| Okumura et al. | Identification of a bone marrow-derived mesenchymal progenitor cell subset that can contribute to the gastric epithelium | |
| Kim et al. | Effect of partial hepatectomy on in vivo engraftment after intravenous administration of human adipose tissue stromal cells in mouse | |
| CN104845932A (zh) | 淫羊藿苷的新用途 | |
| WO2008035908A1 (en) | Therapeutic cell medicine comprising skin tissue derived stem cell | |
| CN114540298A (zh) | 一种干细胞无血清培养基及其制备方法 | |
| JP5388297B2 (ja) | アディポクラスター | |
| CN113106057B (zh) | 一种用于肝细胞移植的自体干细胞制剂及制备方法 | |
| TW201000110A (en) | Method of differentiating mammalian progenitor cells into insulin producing pancreatic islet cells | |
| CN117959337A (zh) | 口腔来源间充质干细胞外泌体在制备预防和治疗肝脏疾病药物中的应用 | |
| CN115125192B (zh) | 一种骨髓上清液及其在细胞培养中的应用 | |
| CN117305236A (zh) | 脂肪间充质干细胞低氧气氛培养及其治疗卵巢早衰的用途 | |
| Bin et al. | Myogenic differentiation of mesenchymal stem cells for muscle regeneration in urinary tract | |
| CN107083367B (zh) | 培养基及其用途与由尿液细胞制备间充质干细胞的方法 | |
| Ma et al. | Identification and characterization of pulmonary mesenchymal stem cells derived from rat fetal lung tissue | |
| CN110628712B (zh) | 基于诱导多能干细胞的治疗级间充质干细胞制备方法及应用 | |
| CN109385399A (zh) | 一种羊水间充质干细胞分化为神经干细胞的方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |