CN112442481A - 脂肪干细胞协同脐带血干细胞分化的方法 - Google Patents
脂肪干细胞协同脐带血干细胞分化的方法 Download PDFInfo
- Publication number
- CN112442481A CN112442481A CN202011360830.7A CN202011360830A CN112442481A CN 112442481 A CN112442481 A CN 112442481A CN 202011360830 A CN202011360830 A CN 202011360830A CN 112442481 A CN112442481 A CN 112442481A
- Authority
- CN
- China
- Prior art keywords
- stem cells
- cells
- cord blood
- adipose
- stem cell
- 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.)
- Granted
Links
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 93
- 210000004700 fetal blood Anatomy 0.000 title claims abstract description 63
- 238000000034 method Methods 0.000 title claims abstract description 27
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 30
- 230000014509 gene expression Effects 0.000 claims abstract description 29
- 210000003958 hematopoietic stem cell Anatomy 0.000 claims abstract description 26
- 210000003743 erythrocyte Anatomy 0.000 claims abstract description 25
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 20
- 230000004069 differentiation Effects 0.000 claims abstract description 19
- 229920001184 polypeptide Polymers 0.000 claims abstract description 18
- 230000035755 proliferation Effects 0.000 claims abstract description 16
- 230000001737 promoting effect Effects 0.000 claims abstract description 12
- 230000006698 induction Effects 0.000 claims abstract description 11
- 108010009307 Forkhead Box Protein O3 Proteins 0.000 claims abstract description 8
- 230000004663 cell proliferation Effects 0.000 claims abstract description 5
- 102000009562 Forkhead Box Protein O3 Human genes 0.000 claims abstract 2
- 210000004027 cell Anatomy 0.000 claims description 50
- 239000006143 cell culture medium Substances 0.000 claims description 21
- 239000012091 fetal bovine serum Substances 0.000 claims description 20
- 239000002609 medium Substances 0.000 claims description 19
- 239000006285 cell suspension Substances 0.000 claims description 14
- 210000003954 umbilical cord Anatomy 0.000 claims description 12
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 10
- 229920002674 hyaluronan Polymers 0.000 claims description 10
- 229960003160 hyaluronic acid Drugs 0.000 claims description 10
- 239000001963 growth medium Substances 0.000 claims description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 7
- 239000012528 membrane Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 229960005322 streptomycin Drugs 0.000 claims description 6
- 238000007664 blowing Methods 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 4
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 3
- 229930182816 L-glutamine Natural products 0.000 claims description 3
- 102000004338 Transferrin Human genes 0.000 claims description 3
- 108090000901 Transferrin Proteins 0.000 claims description 3
- 150000001413 amino acids Chemical group 0.000 claims description 3
- 230000024245 cell differentiation Effects 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
- 229960003957 dexamethasone Drugs 0.000 claims description 3
- 239000012581 transferrin Substances 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims 1
- 238000011160 research Methods 0.000 abstract description 5
- 230000000925 erythroid effect Effects 0.000 abstract description 4
- 101001059929 Caenorhabditis elegans Forkhead box protein O Proteins 0.000 abstract description 3
- 238000012216 screening Methods 0.000 abstract description 2
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 11
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 10
- 102000004127 Cytokines Human genes 0.000 description 8
- 108090000695 Cytokines Proteins 0.000 description 8
- 210000003995 blood forming stem cell Anatomy 0.000 description 8
- 238000003501 co-culture Methods 0.000 description 8
- 108060003196 globin Proteins 0.000 description 8
- 102000018146 globin Human genes 0.000 description 8
- 102100035421 Forkhead box protein O3 Human genes 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 230000007159 enucleation Effects 0.000 description 5
- 230000003394 haemopoietic effect Effects 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 4
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- -1 Aryl Hydrocarbon Chemical class 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 102100031690 Erythroid transcription factor Human genes 0.000 description 3
- 102000007079 Peptide Fragments Human genes 0.000 description 3
- 108010033276 Peptide Fragments Proteins 0.000 description 3
- 210000001185 bone marrow Anatomy 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 108020001507 fusion proteins Proteins 0.000 description 3
- 102000037865 fusion proteins Human genes 0.000 description 3
- 238000002826 magnetic-activated cell sorting Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108010008929 proto-oncogene protein Spi-1 Proteins 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000002195 synergetic effect Effects 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- 102000007469 Actins Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- 101710100588 Erythroid transcription factor Proteins 0.000 description 2
- 208000009329 Graft vs Host Disease Diseases 0.000 description 2
- 101001105486 Homo sapiens Proteasome subunit alpha type-7 Proteins 0.000 description 2
- HYRKAAMZBDSJFJ-LFDBJOOHSA-N Paramethasone acetate Chemical compound C1([C@@H](F)C2)=CC(=O)C=C[C@]1(C)[C@@H]1[C@@H]2[C@@H]2C[C@@H](C)[C@@](C(=O)COC(C)=O)(O)[C@@]2(C)C[C@@H]1O HYRKAAMZBDSJFJ-LFDBJOOHSA-N 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 102100021201 Proteasome subunit alpha type-7 Human genes 0.000 description 2
- 108010087230 Sincalide Proteins 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 102100027654 Transcription factor PU.1 Human genes 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000002738 chelating agent Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 208000024908 graft versus host disease Diseases 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 238000011134 hematopoietic stem cell transplantation Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 229960003966 nicotinamide Drugs 0.000 description 2
- 235000005152 nicotinamide Nutrition 0.000 description 2
- 239000011570 nicotinamide Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000012925 reference material Substances 0.000 description 2
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 238000004114 suspension culture Methods 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 1
- 102100022464 5'-nucleotidase Human genes 0.000 description 1
- PHJPKNUWWHRAOC-PEFMBERDSA-N Asn-Ile-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N PHJPKNUWWHRAOC-PEFMBERDSA-N 0.000 description 1
- APYNREQHZOGYHV-ACZMJKKPSA-N Asp-Cys-Gln Chemical compound C(CC(=O)N)[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC(=O)O)N APYNREQHZOGYHV-ACZMJKKPSA-N 0.000 description 1
- DKQCWCQRAMAFLN-UBHSHLNASA-N Asp-Trp-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(O)=O)C(O)=O DKQCWCQRAMAFLN-UBHSHLNASA-N 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 102100024210 CD166 antigen Human genes 0.000 description 1
- 102100032912 CD44 antigen Human genes 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 102100023126 Cell surface glycoprotein MUC18 Human genes 0.000 description 1
- 108010043471 Core Binding Factor Alpha 2 Subunit Proteins 0.000 description 1
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
- 206010061819 Disease recurrence Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102100037241 Endoglin Human genes 0.000 description 1
- 101150112427 FOXO3 gene Proteins 0.000 description 1
- 108010074860 Factor Xa Proteins 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- UMIRPYLZFKOEOH-YVNDNENWSA-N Glu-Gln-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O UMIRPYLZFKOEOH-YVNDNENWSA-N 0.000 description 1
- QLNKFGTZOBVMCS-JBACZVJFSA-N Glu-Tyr-Trp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O QLNKFGTZOBVMCS-JBACZVJFSA-N 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 102100035716 Glycophorin-A Human genes 0.000 description 1
- 102000006354 HLA-DR Antigens Human genes 0.000 description 1
- 108010058597 HLA-DR Antigens Proteins 0.000 description 1
- 102100021519 Hemoglobin subunit beta Human genes 0.000 description 1
- 108091005904 Hemoglobin subunit beta Proteins 0.000 description 1
- 102100030826 Hemoglobin subunit epsilon Human genes 0.000 description 1
- 108091005879 Hemoglobin subunit epsilon Proteins 0.000 description 1
- 102100030387 Hemoglobin subunit zeta Human genes 0.000 description 1
- 108091005905 Hemoglobin subunit zeta Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000678236 Homo sapiens 5'-nucleotidase Proteins 0.000 description 1
- 101000980840 Homo sapiens CD166 antigen Proteins 0.000 description 1
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 1
- 101000623903 Homo sapiens Cell surface glycoprotein MUC18 Proteins 0.000 description 1
- 101000881679 Homo sapiens Endoglin Proteins 0.000 description 1
- 101001066268 Homo sapiens Erythroid transcription factor Proteins 0.000 description 1
- 101100391196 Homo sapiens FOXO3 gene Proteins 0.000 description 1
- 101001074244 Homo sapiens Glycophorin-A Proteins 0.000 description 1
- 101000935043 Homo sapiens Integrin beta-1 Proteins 0.000 description 1
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 1
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 description 1
- 102100025304 Integrin beta-1 Human genes 0.000 description 1
- MQMIRLVJXQNTRJ-SDDRHHMPSA-N Lys-Gln-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCCCN)N)C(=O)O MQMIRLVJXQNTRJ-SDDRHHMPSA-N 0.000 description 1
- SNIXRMIHFOIVBB-UHFFFAOYSA-N N-Hydroxyl-tryptamine Chemical compound C1=CC=C2C(CCNO)=CNC2=C1 SNIXRMIHFOIVBB-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- ISYSEOWLRQKQEQ-JYJNAYRXSA-N Phe-His-Glu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(O)=O)C(O)=O ISYSEOWLRQKQEQ-JYJNAYRXSA-N 0.000 description 1
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 1
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 1
- 102100025373 Runt-related transcription factor 1 Human genes 0.000 description 1
- 102100022513 Selenocysteine lyase Human genes 0.000 description 1
- 108010017842 Telomerase Proteins 0.000 description 1
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 210000000579 abdominal fat Anatomy 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- YRQNKMKHABXEJZ-UVQQGXFZSA-N chembl176323 Chemical compound C1C[C@]2(C)[C@@]3(C)CC(N=C4C[C@]5(C)CCC6[C@]7(C)CC[C@@H]([C@]7(CC[C@]6(C)[C@@]5(C)CC4=N4)C)CCCCCCCC)=C4C[C@]3(C)CCC2[C@]2(C)CC[C@H](CCCCCCCC)[C@]21C YRQNKMKHABXEJZ-UVQQGXFZSA-N 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 229910001431 copper ion Inorganic materials 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 108091000099 cysteine desulfurase Proteins 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
- 239000011325 microbead Substances 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000007026 protein scission Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 238000005406 washing Methods 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
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0641—Erythrocytes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- 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
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
-
- 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
-
- 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
- C12N2501/105—Insulin-like growth factors [IGF]
-
- 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
- C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
-
- 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
- C12N2501/14—Erythropoietin [EPO]
-
- 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
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2303—Interleukin-3 (IL-3)
-
- 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
- C12N2501/90—Polysaccharides
- C12N2501/905—Hyaluronic acid
-
- 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
- C12N2501/998—Proteins not provided for elsewhere
-
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
- C12N2502/1352—Mesenchymal stem cells
- C12N2502/1382—Adipose-derived stem cells [ADSC], adipose stromal stem cells
-
- 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1369—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from blood-borne mesenchymal stem cells, e.g. MSC from umbilical blood
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Rheumatology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及一种脂肪干细胞协同脐带血干细胞分化的方法。本发明通过筛选和优化获得能够特异性促进脐带血干细胞中FOXO3高表达的促进活性肽，该多肽能够通过提高FOXO3的表达，来促进脐带血干细胞的增殖以及向红细胞的分化；还提供了一种改进的采用脂肪干细胞来提高脐带血干细胞增殖的方法，所述方法包括将脂肪干细胞与脐带血干细胞一起进行培育而获得。将扩增的造血干细胞进一步的在特制的诱导培养基中培养能够获得分化的红细胞。本发明能够高速度的扩增造血干细胞并且能够获得红系特性较好的红细胞，可以大规模用于后续的临床研究，具有较好的应用价值。
Description
技术领域
本发明涉及生物医药领域，更具体的，涉及一种脂肪干细胞协同脐带血干细胞分化的方法。
背景技术
脐带血作为一种新的造血干细胞移植的供体，具有来源广泛、易于采集、对供体无伤害、HLA配型要求低、造血干/祖细胞(HSPC)含量高等优点。而且，脐带血的制备时间相比于从一个匹配的无关供者体内获得造血干细胞所需的时间要短4-5周。此外，脐带血移植(UCBT)后的病情复发率和移植物抗宿主病的发病率相比于其他来源的造血干细胞移植也是较低的。然而，UCBT应用于临床最大的局限就是脐带血中可提取到的总的有核细胞(TNC)数量过少。研究表明： HLA匹配的位点越少，需要TNC的量就越多。根据疾病的类型，单份充足的脐带血数量被定义为：HLA位点全相合需>3×107TNC/kg，5个位点相合需>4× 107TNC/kg，4个位点相合则需>5×107TNC/kg。脐带血中所含的有核细胞数，仅有外周血和骨髓中可用有核细胞浓度的5％-10％，如若进行低数量的脐带血移植，会引起中性粒细胞恢复延迟并增加细菌及病毒的感染风险。而双份脐带血移植，虽然扩大了成人使用脐带血进行移植的范围，但会增加移植物抗宿主病的发病率，延长血小板恢复时间，从而引发一系列问题。
为了解决脐带干细胞的扩增问题，目前已有单独添加细胞因子扩增脐带血。许多研究仅证实了，不同的细胞因子在HSC扩增过程中具有协同作用。脐带血经过细胞因子的扩增，虽然有核细胞及祖细胞的数量有所增加，细胞表面表达的相关分子能够促进细胞的归巢作用，但细胞发生了不同程度的分化。因为，HSC在体内是依赖于局部微环境以及包括转录因子、细胞周期调控因子、生长因子和黏附分子在内的复杂的内部和外部信号的严格调控，来维持自我更新和多向分化之间的平衡。因此，体外扩增很难维持HSC原有的干性。大部分研究实验也证实了体外扩增的脐带血HSPC长期植入能力较差，而短期内脐带血HSPC比例占据植入细胞的主导地位。从目前的研究来看，用细胞因子扩增脐带血，虽然TNC的总量有明显的增加，但因扩增的同时诱导了早期祖细胞分化成归巢能力较弱的细胞群，而使UCBT的成功率没有得到提高。所以科学家们利用一些特异性小分子来阻止脐带血体外扩增时早期祖细胞的分化，使扩增的细胞减少分化，能够拥有更好的骨髓归巢能力。例如铜离子螯合剂CopperChelation(TEPA，StemEx)、 NotchLigand、烟酰胺(Nicotinamide)、ArylHydrocarbon Antagonist (StemRegenin1，SR1)等。很多小分子扩增HSPC的试验都正在进行多中心的临床试验，其中StemEx已完成III期试验。
干细胞微环境是干细胞生长的微环境，其在维持HSC自我更新及多向分化中发挥重要作用。而通过模拟造血微环境促进HSC的体外扩增技术也日益成熟。MSC 对HSC就拥有重要的造血支持作用，主要因为：MSC能够提供多种体外扩增的信号通路，这些信号通路在基于单独利用细胞因子来进行HPC悬浮培养中是没有的。MSC可通过直接接触或分泌内源性细胞因子等方式调控HSC。而且，利用MSC 滋养层培养扩增HSPC可避免CD34及CD133分选造成的细胞数的减少。利用骨髓间充质干细胞(BM-MSC)作为饲养层扩增脐带血已经完成了I/II期的临床试验，该试验将脐带血与贴壁的BM-MSC、造血相关细胞因子共培养7d，然后单独使用细胞因子再扩增培养7d。最终实现脐带血HSPC中CD34+细胞数目中位值增长为原有细胞中位数目的30倍。
但是目前，针对采用脂肪干细胞来提高脐带血干细胞的增殖和红细胞分化的研究还没有。
发明内容
本发明提供一种改进的采用脂肪干细胞来提高脐带血干细胞的增殖和红细胞分化的方法。
本发明人前期筛选获得了能够特异性促进脐带血干细胞中FOXO3高表达的两个促进活性肽。这两个多肽能够通过提高FOXO3的表达，来促进脐带血干细胞的增殖以及向红细胞的分化。
另外一方面，还提供一种改进的采用脂肪干细胞来提高脐带血干细胞增殖的方法，所述方法包括将脂肪干细胞与脐带血干细胞一起进行培育而获得。
更进一步的，所述促进增殖的步骤包括，取24孔Transwell培养板一块，用无菌镊子小心夹出含半透膜的上室，向其下室中加入用脂肪干细胞培养基重悬后的脂肪干细胞悬液吹打混匀，用移液枪小心向上室中加入脐带血干细胞细胞悬液，使得上室含细胞：下室含细胞含量之比为1:1-1:10。其中脐带造血干细胞培养基：DMEM-F12培养基90％+FBS 10％+SEQ ID NO：1多肽50μg/mL +SCF15ng/ml+IL-330ng/ml+透明质酸10μg/mL。脂肪干细胞培养基：DMEM-F12 培养基44.5ml+胎牛血清(FBS)5ml+青-链霉素500ul混匀。
更进一步的，本发明提供一种改进的采用脂肪干细胞来提高脐带血干细胞增殖并分化为红细胞的方法，所述方法包括以下步骤：
(1)增殖的步骤：取24孔Transwell培养板一块，用无菌镊子小心夹出含半透膜的上室，向其下室中加入用脂肪干细胞培养基重悬后的脂肪干细胞悬液吹打混匀，用移液枪小心向上室中加入脐带血干细胞细胞悬液，使得上室含细胞：下室含细胞含量之比为1:1-1:10。其中脐带造血干细胞培养基：DMEM-F12培养基90％+FBS 10％+SEQ ID NO：1多肽50μg/mL+SCF15ng/ml+IL-330ng/ml+透明质酸10μg/mL。脂肪干细胞培养基：DMEM-F12培养基44.5ml+胎牛血清(FBS) 5ml+青-链霉素500ul混匀；
(2)分化的步骤：将步骤(1)扩增获得的造血干细胞离心后收获细胞，继续在诱导培养基中进行继续红细胞分化8d后，收获相应的分化红细胞；所述的诱导培养基组成为：以StemSpan SFEM为基础培养基，其中添加终浓度分别为 SEQ ID NO：1多肽50ng/ml，SCF400ng/ml，IGF-160ng/ml，Epo 15IU/ml，转铁蛋白100μg/ml，透明质酸10μg/mL，L-谷氨酰胺1mM，地塞米松1.5μM。
对于本发明的肽，提供序列号1中的氨基酸序列肽。
并提供一种用于促进脐带血干细胞增殖的培养基，所述培养基组成为： DMEM-F12培养基90％+FBS 10％+SEQ ID NO：1多肽50μg/mL+SCF15ng/ml+IL-3 30ng/ml+透明质酸10μg/mL。
以上述SEQ ID NO：1氨基酸序列表达的肽，可通过在该领域广泛公知的多种方法获得。详细说明：利用基因重组和蛋白质表达系统制造，或通过肽合成等化学合成在试管内合成的方法，以及无细胞蛋白质合成法等。更具体地说，可以由该领域众所周知的方法如自动肽合成器合成，也可以由转基因技术生产，但并不局限于此。例如，通过转基因制备融合伙伴与本发明的肽段融合蛋白编码的融合基因，并用它将宿主微生物进行性状转化，然后在宿主微生物中以融合蛋白的形式表达出来，然后将蛋白质利用裂解酶或化合物，可以从融合蛋白中切割分离本发明的肽段，从而生产出想要的肽段。为此，融合伙伴和本发明的肽基因之间，例如Factor Xa或端粒酶等蛋白裂解酶CNBr或羟色胺，编码可能被化合物切割的氨基酸残基的DNA序列。可以插入。
用本发明的上述SEQ ID NO：1的氨基酸序列表达的肽，也可以包括为增加靶化序列标签(tag)标记的残基半衰期或肽稳定性的特定目的而设计的附加氨基酸序列。
有益效果
本发明通过筛选和优化获得能够特异性促进脐带血干细胞中FOXO3高表达的两个促进活性肽。这两个多肽能够通过提高FOXO3的表达，来促进脐带血干细胞的增殖以及向红细胞的分化；还提供了一种改进的采用脂肪干细胞来提高脐带血干细胞增殖的方法，所述方法包括将脂肪干细胞与脐带血干细胞一起进行培育而获得。将扩增的造血干细胞进一步的在特制的诱导培养基中培养能够获得分化的红细胞。本发明能够高速度的扩增造血干细胞并且能够获得红系特性较好的红细胞，可以大规模用于后续的临床研究，具有较好的应用价值。
附图说明
图1：FOXO3表达量结果图
图2：细胞生长曲线图
图3：细胞因子表达水平结果图
图4：细胞脱核水平结果图
具体实施方式
下面结合具体实施例介绍本发明的技术方案。下述实施例中未特别强调的实验材料均为常规实验材料，无特别要求，都是本领域技术人员容易获得的常规材料。
实施例1脐带造血干细胞的分离
首先用Ficoll淋巴细胞分离液从脐带血中分离出单个核细胞，加100μL FcR封闭试剂和100μL CD34免疫微珠到细胞悬液中，混匀，放入冰箱，4℃孵育30min。用MACS液冲洗进行洗脱，去除细胞碎片和杂细胞等。取下柱子放在收集管上，加入MACS液(LS柱：5ml)，接上柱塞，用力快速推下冲洗出吸附的细胞。重复进行过柱一次，之后离心力300g，离心10min去上清，500μL MACS 液重悬细胞。收集细胞，进行细胞计数，流式细胞仪进行CD34细胞纯度测定。经MidiMACS免疫磁珠法纯化的人脐带血造血干细胞经流式细胞仪检测，CD34细胞的百分比为(93.2±4.6)％，符合造血干细胞的特征，备用。
实施例2多肽在促进FOXO3表达中的作用
将SEQ ID NO：1的多肽分别按照0、1ug/mL、10ug/mL、50ug/mL、100ug/mL 的浓度剂量加入到StemSpan SFEM培养基中。将实施例1分离得到的细胞按照 5×105cells/ml的密度接种于1ml培养基悬浮培养48h后进行FOXO3基因表达量检测。提取相同细胞量的RNA，同时TaKaRa试剂盒将RNA反转录成cDNA。以下述引物进行RT-PCR检测。FOXO3引物序列为：上游5′ -CGGACAAACGGCTCACTCT-3′，下游5′-GGACCCGCATGAATCGACTAT-3′；GAPDH引物序列为：上游5′-ACAACTTTGGTATCGTGGAAGG-3′，下游5′ -GCCATCACGCCACAGTTTC-3′。置于7300HTRT-PCR仪进行扩增。反应条件按TaKaRa 试剂盒条件设置。利用ABI7300系统SDSSoftware软件进行分析，使用2-ΔΔCt方法计算。实验均重复3次。结果如图1所示。
在使用了多肽处理后，FOXO3的基因表达量得到了显著的提高。具体的，在浓度为100ug/mL时候其促进基因表达量提高到(3.4±0.3)倍。
实施例3脂肪干细胞的分离
将健康人来源的腹部脂肪剪成0.1cm*0.1cm*0.1cm大小组织，用PBS缓冲液冲洗3-5次。以5倍脂肪体积的胶原酶I消化，反复将脂肪组织剪碎至糊状后， 37℃摇晃均匀30min，用250ul尼龙纱网将消化后的脂肪组织过滤入离心管中， 1000r/min离心30min，弃去上清液和悬浮的脂肪碎块，沉淀重悬于00.075mmol/L 氯化钾，静置10min后离心，弃去伤情，沉淀重悬于含10％胎牛血清的DMEM培养液中，移入培养皿中，置于37℃，5％CO2温箱，以100％湿度进行培养，隔天更换培养液，待细胞铺满90％培养皿底部面积后，以0.25％胰蛋白酶消化，按1:3 的比例进行传代。取第三代细胞，用流式细胞仪检测标记物的表达情况。结果显示，标记物CD90,CD105,CD44,CD73,CD146,CD166及CD29均高表达，表达率高于96％；标记物CD45,CD31,CD34及HLA-DR均低表达；实验结果表明，脂肪干细胞分离成功。
实施例4脂肪干细胞对脐带造血干细胞的促进作用
(1)共培养实验组：取24孔Transwell培养板一块，用无菌镊子小心夹出含半透膜的上室，向其下室中加入600ul用脂肪干细胞培养基重悬后的实施例3 分离的脂肪干细胞悬液吹打混匀。用移液枪小心向上室中加入100ul实施例1 分离的脐带血干细胞细胞悬液。使得每孔上室含细胞104个,下室含细胞5×104个。每孔总液体量700ul，设置3个复孔。其中脐带造血干细胞培养基：DMEM-F12培养基90％+FBS 10％+SEQ ID NO：1多肽50μg/mL+SCF15ng/ml+IL-330ng/ml+透明质酸10μg/mL。脂肪干细胞培养基：DMEM-F12培养基44.5ml+胎牛血清(FBS) 5ml+青-链霉素500ul混匀。
(2)共培养无肽实验组：取24孔Transwell培养板一块，用无菌镊子小心夹出含半透膜的上室，向其下室中加入600ul用脂肪干细胞培养基重悬后的实施例3分离的脂肪干细胞悬液吹打混匀。用移液枪小心向上室中加入100ul实施例 1分离的脐带血干细胞细胞悬液。使得每孔上室含细胞104个,下室含细胞5×104个。每孔总液体量700ul，设置3个复孔。其中脐带造血干细胞培养基：DMEM-F12 培养基90％+FBS 10％+SCF15ng/ml+IL-330ng/ml+透明质酸10μg/mL。脂肪干细胞培养基：DMEM-F12培养基44.5ml+胎牛血清(FBS)5ml+青-链霉素500ul混匀。
(3)脐带造血干单独培养组：取实施例1分离的脐带造血干细胞加入24孔板中，设置3个复孔，再向每孔中加入700ul脐带造血干细胞培养基，吹打混匀，使每孔含细胞104个，液体总量700ul/孔。脐带造血干细胞培养基：DMEM-F12培养基90％+FBS 10％+SCF15ng/ml+IL-330ng/ml+透明质酸10μg/mL。
上述3组分别培养1、3、5、7天后CCK-8法检测脐带血干细胞的增殖情况。收集3组的脐带血干细胞样本，将收集到的EP管中的脐带血干细胞样本标记后，置于离心机中，1200rpm离心5min，弃去上清液，重悬至100ul/管。将每个EP 管中的重悬后的脐带血干细胞悬液吸出分别置于96孔板中，100ul/孔，标记每孔细胞来源的组。取100ul不含细胞的脐带血干细胞培养基于96孔板中，设置 3个复孔，作为对照。避光条件下，向每孔加入CCK-8试剂10ul，于37℃二氧化碳敷箱中孵育5小时，取出并打开孔板盖子，放入使用酶标仪测定每孔在450 nm处的OD值。每组3个复孔，实验重复3次。绘制HSC增殖曲线。结果如图3 所示。
从生长曲线是看出各组脐带血干细胞数量均随培养时间的延长而增加，第3天开始进入对数生长期，与脂肪干细胞共培养对脐带干细胞增殖有促进作用，特别是添加了多肽之后的促进效果更加显著，在第7天，OD达到了3.5±0.3，具有较好的增殖效果。
实施例5增殖后的造血干细胞向红细胞分化
将实施例4三组试验后获得造血干细胞离心后收获细胞，继续在诱导培养基中进行继续红细胞分化8d后，收获相应的分化红细胞进行鉴定。所述的诱导培养基组成为：以StemSpan SFEM为基础培养基，其中添加终浓度分别为SEQ ID NO： 1多肽50ng/ml，SCF400ng/ml，IGF-160ng/ml，Epo 15IU/ml，转铁蛋白100μg/ml， L-谷氨酰胺1mM，地塞米松1.5μM。
将所述细胞提取RNA，反转录为cDNA，用RT-PCR检测造血干细胞的标志物CD34及红系发育相关转录因子PU.1、Run X1、GATA1、SCL、和珠蛋白ζ、珠蛋白ε、珠蛋白β的表达情况，以β-actin(对照)的表达情况。采用如图3 所示的引物组进行RT-PCR鉴定。所述PCR的条件为95℃7min→(94℃50s→58℃ 30s→72℃1min)×30循环→72℃10min。具体的引物序列如下所示。
GATA-1引物：F：CAGGTACTGCCCATCTCTAC；R:TCTGGCTACAAGAGGAGAAG；SCL 引物:F：GCTGGCTTTTCTGTTTCCTG；R:TGACAACCCCAGGTCTTAGG；PU.1引物： F:CGACCATTACTGGGACTTCC；R:TTCTTCTTCACCTTCTTGACC；RUNX1引物： F:ATGTGGTCCTATT TAAGCCAGCCC；R:TCATCTGGCTGAAGACACCAGCTT；CD34引物： F:AAATXCCTCTTCCTCTGAGGCTGGA；R:AAGAGGCAGCTGGTGATAAGGGTT；β-globin引物：F：GGGCAGGTTGGTATCAAGGTTAC；R：GGGGAAAGAAAACATCAAGCG；ε-globin引物： F：AAGATGAATGTGGAAGAGGCTG；R：TTAGCAAAGGCGGGCTTGAG；ζ-globin引物：F：CCAAGACTGAGAGGACCATCATTG；R：AGGACAGAGGATACGACCGATAGG；β-Actin：F： GATCCACATCTGCTGGAAGG；R:AAGTGTGACGTTGACATCCG。以β-actin的表达量作为相对表达量基础，结果如图4所示。
结果显示，随着培养时间的延长，造血干细胞基因CD34的表达逐渐降低，表明随培养时间的延长，细胞逐渐向成熟分化。共培养实验组的CD34表达降低最快，表明多肽能够快速的促进造血干细胞向红细胞分化，特别是在共培养组， CD34的表达量只有3±0.5，比共培养无肽实验组的10±1.0要低的多，表明多肽能够促进造血干细胞分化为红细胞。此外本申请实验组早期造血相关转录因子 SCL的表达持续为阴性，表明诱导体系内的细胞已经发生分化。在实验组，PU.1 随着诱导时间的延长表达逐渐减弱，而GATA-1一直持续高表达，诱导体系内的细胞随向红系分化。此外，胚胎期珠蛋白ζ表达逐渐减弱，成体珠蛋白β持续表达，表明培养体系内红细胞正在经历珠蛋白表达由胚胎期模式向成体模式的转变，红细胞逐渐成熟分化。特别是共培养实验组的成体珠蛋白β表达量比共培养无肽实验组的表达量提高了1.42倍。
实施例6红细胞脱核情况的分析：
将实施例5三组分化得到的红细胞，采用流式细胞术分析CD235a阳性 /Hoechst33342阴性细胞的比例，该比例可以反映细胞脱核情况。结果如图4 所示。
由图可知，将实施例5三组分化得到的红细胞，特别是共培养实验组细胞脱核水平可以达到88％的水平，比现有技术的脱核水平得到显著的提高。
应当理解的是，本发明在其应用上并不一定局限于在以下说明中所描述和/ 或在附图中所说明的组件的构造和布置的细节。本发明能够具有除所述和以不同方式实践或进行的那些实施方案之外的实施方案。而且，应理解本文所采用的短语和术语以及摘要出于描述目的并且不应视为限制性的。
序列表
<110> 北京广未生物科技有限公司
<120> 脂肪干细胞协同脐带血干细胞分化的方法
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Glu Gln Ile Asp Cys Gln Asn Ile Gln Glu Tyr Trp Asp Trp Asp Ser
1 5 10 15
Phe His Glu Lys Gln Pro Asp Thr
20
Claims (4)
1.一种改进的采用脂肪干细胞来提高脐带血干细胞增殖并分化为红细胞的方法，所述方法包括以下步骤：
(1)增殖的步骤：取24孔Transwell培养板一块，用无菌镊子小心夹出含半透膜的上室，向其下室中加入用脂肪干细胞培养基重悬后的脂肪干细胞悬液吹打混匀，用移液枪小心向上室中加入脐带血干细胞细胞悬液，使得上室含细胞：下室含细胞含量之比为1:1-1:10；其中脐带造血干细胞培养基：DMEM-F12培养基90％+FBS 10％+SEQ ID NO：1多肽50μg/mL+SCF15ng/ml+IL-3 30ng/ml+透明质酸10μg/mL；脂肪干细胞培养基：DMEM-F12培养基44.5ml+胎牛血清(FBS)5ml+青-链霉素500ul混匀；
(2)分化的步骤：将步骤(1)扩增获得的造血干细胞离心后收获细胞，继续在诱导培养基中进行继续红细胞分化8d后，收获相应的分化红细胞；所述的诱导培养基组成为：以StemSpan SFEM为基础培养基，其中添加终浓度分别为SEQ ID NO：1多肽50ng/ml，SCF400ng/ml，IGF-1 60ng/ml，Epo 15IU/ml，转铁蛋白100μg/ml，透明质酸10μg/mL，L-谷氨酰胺1mM，地塞米松1.5μM。
2.一种改进的采用脂肪干细胞来提高脐带血干细胞增殖的方法，所述方法包括将脂肪干细胞与脐带血干细胞一起进行培育而获得；在培育过程中添加了权利要求1所述的促进活性肽。
3.一种改进的采用脂肪干细胞来提高脐带血干细胞增殖的方法，其特征在于所述促进增殖的步骤包括，取24孔Transwell培养板一块，用无菌镊子小心夹出含半透膜的上室，向其下室中加入用脂肪干细胞培养基重悬后的脂肪干细胞悬液吹打混匀，用移液枪小心向上室中加入脐带血干细胞细胞悬液，使得上室含细胞：下室含细胞含量之比为1:1-1:10。其中脐带造血干细胞培养基：DMEM-F12培养基90％+FBS 10％+SEQ ID NO：1多肽50μg/mL+SCF15ng/ml+IL-3 30ng/ml+透明质酸10μg/mL；脂肪干细胞培养基：DMEM-F12培养基44.5ml+胎牛血清(FBS)5ml+青-链霉素500ul混匀。
4.一种能够特异性促进脐带血干细胞中FOXO3高表达的促进活性肽，其氨基酸序列如SEQ ID NO：1所示。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011360830.7A CN112442481B (zh) | 2020-11-27 | 2020-11-27 | 脂肪干细胞协同脐带血干细胞分化的方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011360830.7A CN112442481B (zh) | 2020-11-27 | 2020-11-27 | 脂肪干细胞协同脐带血干细胞分化的方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112442481A true CN112442481A (zh) | 2021-03-05 |
| CN112442481B CN112442481B (zh) | 2021-09-17 |
Family
ID=74737833
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011360830.7A Active CN112442481B (zh) | 2020-11-27 | 2020-11-27 | 脂肪干细胞协同脐带血干细胞分化的方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112442481B (zh) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1596303A (zh) * | 2001-11-09 | 2005-03-16 | 阿特塞尔科学公司 | 利用基质细胞支持胚胎干细胞和成体干细胞的方法和组合物 |
| KR101017710B1 (ko) * | 2009-02-05 | 2011-02-25 | 한양대학교 산학협력단 | 세포 스페로이드 형태의 이식용 성체 줄기세포 집합체 및 그 제조방법 |
| CN105039257A (zh) * | 2015-07-24 | 2015-11-11 | 深圳爱生再生医学科技有限公司 | 造血干细胞的体外扩增培养方法 |
-
2020
- 2020-11-27 CN CN202011360830.7A patent/CN112442481B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1596303A (zh) * | 2001-11-09 | 2005-03-16 | 阿特塞尔科学公司 | 利用基质细胞支持胚胎干细胞和成体干细胞的方法和组合物 |
| KR101017710B1 (ko) * | 2009-02-05 | 2011-02-25 | 한양대학교 산학협력단 | 세포 스페로이드 형태의 이식용 성체 줄기세포 집합체 및 그 제조방법 |
| CN105039257A (zh) * | 2015-07-24 | 2015-11-11 | 深圳爱生再生医学科技有限公司 | 造血干细胞的体外扩增培养方法 |
Non-Patent Citations (2)
| Title |
|---|
| SOFIA BERGLUND等: "Advances in umbilical cord blood cell therapy: the present and the future", 《EXPERT OPINION BIOLOGICAL THERAPY》 * |
| 宋克东等: "脐血造血干/祖细胞与脂肪干细胞共培养的作用距离研究", 《高校化学工程学报》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112442481B (zh) | 2021-09-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR100489248B1 (ko) | 제대혈 유래 간엽줄기세포ㆍ전구세포의 분리배양방법 및간엽조직으로의 분화유도방법 | |
| KR101211913B1 (ko) | 양막유래 중간엽 줄기세포 배양을 위한 배지조성물 및 이를 이용한 양막유래 중간엽 줄기세포의 배양방법 | |
| CN102876630B (zh) | 一种高效分离和扩增人脐带血间充质干细胞的方法 | |
| US20110236978A1 (en) | Reprogramming cells toward a pluripotent state | |
| CN112662627B (zh) | 多能干细胞分化为自然杀伤细胞的培养液及分化方法 | |
| CN112481207B (zh) | 一种脂肪干细胞在促进脐带血造血干细胞增殖的方法 | |
| CN111826348A (zh) | 一种人诱导性多能干细胞来源的间充质干细胞体外高效制备方法和应用 | |
| CN107418930B (zh) | 一种纯化与扩增人骨髓间充质干细胞的制备方法 | |
| CN110872574B (zh) | 一种高效可靠的hESC-MSC制备方法 | |
| CN104726401A (zh) | 一种利用胎盘间充质干细胞提高脐血间充质干细胞培养成功率的方法 | |
| CN111235100B (zh) | 一种人脐带血间充质干细胞的培养方法 | |
| WO2013102114A1 (en) | Manufacturing process for fresh and frozen stem cells | |
| CN109897815B (zh) | 一种无需包被的脂肪内皮祖细胞的高效分离和培养方法 | |
| CN115125192A (zh) | 一种骨髓上清液及其在细胞培养中的应用 | |
| CN112442481B (zh) | 脂肪干细胞协同脐带血干细胞分化的方法 | |
| CN113750220B (zh) | 间充质干细胞联合tpo及其类似物在治疗慢性髓性白血病中的应用 | |
| US20240294878A1 (en) | Method of producing exosomes from immortalized clonal mesenchymal stem cells (hmscs) derived from hla homozygous human induced pluripotent stem cells (hipsc's) derived from cord blood | |
| US20240293470A1 (en) | EXOSOMES DERIVED FROM IMMORTALIZED MESENCHYMAL STROMAL CELLS (hMSCs) FOR USE AS A MEDICAMENT | |
| CN106434546B (zh) | 完全利用脐带资源制备间充质干细胞的方法 | |
| CN116218772A (zh) | 一种尿源混合细胞培养方法 | |
| WO2023285529A1 (en) | Method of producing immortalized clonal mesenchymal stem cells (hmscs) from hla homozygous human induced pluripotent stem cells (hipsc's) derived from cord blood | |
| WO2008070258A2 (en) | Directed differentiation of human embryonic stem cells into mesenchymal/stromal cells | |
| CN114058573B (zh) | 一种含有生物素的培养基 | |
| EP4541882A1 (en) | Thymic epithelial cell production method | |
| CN114517178B (zh) | Trolox在延缓间充质干细胞衰老中的应用 |
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 | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20210730 Address after: 450000 floor 1, building 6, phase I, Zhongyuan Financial Industrial Park, Mingli road and Baifo South Road, Zhengzhou area (Zhengdong), Henan pilot Free Trade Zone, Zhengzhou City, Henan Province Applicant after: Henan Beike Biotechnology Co.,Ltd. Address before: 701-286, 7th floor, building 1, guangyuanzha 5, Haidian District, Beijing 100081 Applicant before: Beijing Guangwei Biotechnology Co., Ltd |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |