CN106282100B - A method of the purifying tissue-derived Preadipocyte In Vitro of ox fetal skeletal muscle - Google Patents
A method of the purifying tissue-derived Preadipocyte In Vitro of ox fetal skeletal muscle Download PDFInfo
- Publication number
- CN106282100B CN106282100B CN201610740549.3A CN201610740549A CN106282100B CN 106282100 B CN106282100 B CN 106282100B CN 201610740549 A CN201610740549 A CN 201610740549A CN 106282100 B CN106282100 B CN 106282100B
- Authority
- CN
- China
- Prior art keywords
- cell
- added
- sorting
- oil red
- tissue
- 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
- 210000002027 skeletal muscle Anatomy 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 20
- 238000000338 in vitro Methods 0.000 title claims abstract description 13
- 210000001519 tissue Anatomy 0.000 title claims abstract description 13
- 210000000229 preadipocyte Anatomy 0.000 title claims abstract description 9
- 230000001605 fetal effect Effects 0.000 title claims abstract description 7
- 210000004027 cell Anatomy 0.000 claims abstract description 102
- 210000003205 muscle Anatomy 0.000 claims abstract description 26
- 239000000872 buffer Substances 0.000 claims abstract description 25
- 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 claims abstract description 22
- 210000003754 fetus Anatomy 0.000 claims abstract description 9
- 102000009465 Growth Factor Receptors Human genes 0.000 claims abstract description 8
- 108010009202 Growth Factor Receptors Proteins 0.000 claims abstract description 8
- 230000000702 anti-platelet effect Effects 0.000 claims abstract description 7
- 239000003146 anticoagulant agent Substances 0.000 claims abstract description 7
- 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 abstract description 7
- 229960003957 dexamethasone Drugs 0.000 claims abstract description 7
- APIXJSLKIYYUKG-UHFFFAOYSA-N 3 Isobutyl 1 methylxanthine Chemical compound O=C1N(C)C(=O)N(CC(C)C)C2=C1N=CN2 APIXJSLKIYYUKG-UHFFFAOYSA-N 0.000 claims abstract description 4
- 101001011741 Bos taurus Insulin Proteins 0.000 claims abstract description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 4
- IXIBAKNTJSCKJM-BUBXBXGNSA-N bovine insulin Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)C(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 IXIBAKNTJSCKJM-BUBXBXGNSA-N 0.000 claims abstract description 4
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 4
- 239000012894 fetal calf serum Substances 0.000 claims abstract description 4
- 239000000463 material Substances 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 19
- 239000008363 phosphate buffer Substances 0.000 claims description 19
- 239000001963 growth medium Substances 0.000 claims description 14
- 239000006228 supernatant Substances 0.000 claims description 12
- 239000000725 suspension Substances 0.000 claims description 9
- 230000006698 induction Effects 0.000 claims description 8
- 230000010261 cell growth Effects 0.000 claims description 7
- 102000001393 Platelet-Derived Growth Factor alpha Receptor Human genes 0.000 claims description 6
- 108010068588 Platelet-Derived Growth Factor alpha Receptor Proteins 0.000 claims description 6
- 230000009977 dual effect Effects 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
- 238000012360 testing method Methods 0.000 claims description 5
- 230000002293 adipogenic effect Effects 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 235000013372 meat Nutrition 0.000 claims description 4
- 102000029816 Collagenase Human genes 0.000 claims description 3
- 108060005980 Collagenase Proteins 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 108060003951 Immunoglobulin Proteins 0.000 claims description 3
- 102000004877 Insulin Human genes 0.000 claims description 3
- 108090001061 Insulin Proteins 0.000 claims description 3
- 229930040373 Paraformaldehyde Natural products 0.000 claims description 3
- 240000004922 Vigna radiata Species 0.000 claims description 3
- 235000010721 Vigna radiata var radiata Nutrition 0.000 claims description 3
- 235000011469 Vigna radiata var sublobata Nutrition 0.000 claims description 3
- 230000002776 aggregation Effects 0.000 claims description 3
- 238000004220 aggregation Methods 0.000 claims description 3
- 210000001691 amnion Anatomy 0.000 claims description 3
- 238000004113 cell culture Methods 0.000 claims description 3
- 229960002424 collagenase Drugs 0.000 claims description 3
- 210000000805 cytoplasm Anatomy 0.000 claims description 3
- 230000029087 digestion Effects 0.000 claims description 3
- 239000000975 dye Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 102000018358 immunoglobulin Human genes 0.000 claims description 3
- 229940125396 insulin Drugs 0.000 claims description 3
- 229920002866 paraformaldehyde Polymers 0.000 claims description 3
- 238000010186 staining Methods 0.000 claims description 3
- 108091008606 PDGF receptors Proteins 0.000 claims 1
- 102000011653 Platelet-Derived Growth Factor Receptors Human genes 0.000 claims 1
- 239000002609 medium Substances 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 abstract 1
- 210000000130 stem cell Anatomy 0.000 description 18
- 210000001789 adipocyte Anatomy 0.000 description 16
- 210000001057 smooth muscle myoblast Anatomy 0.000 description 15
- 241001465754 Metazoa Species 0.000 description 14
- 238000011160 research Methods 0.000 description 14
- 239000000835 fiber Substances 0.000 description 13
- 102000000536 PPAR gamma Human genes 0.000 description 12
- 108010016731 PPAR gamma Proteins 0.000 description 12
- 230000000694 effects Effects 0.000 description 12
- 230000004069 differentiation Effects 0.000 description 10
- 210000001671 embryonic stem cell Anatomy 0.000 description 10
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- 102000015735 Beta-catenin Human genes 0.000 description 7
- 108060000903 Beta-catenin Proteins 0.000 description 7
- 238000011161 development Methods 0.000 description 7
- 230000018109 developmental process Effects 0.000 description 7
- 230000012010 growth Effects 0.000 description 7
- 238000007918 intramuscular administration Methods 0.000 description 7
- 210000000107 myocyte Anatomy 0.000 description 7
- 241000894007 species Species 0.000 description 7
- 230000011759 adipose tissue development Effects 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 230000004913 activation Effects 0.000 description 5
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 5
- 108020003175 receptors Proteins 0.000 description 5
- 102000005962 receptors Human genes 0.000 description 5
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 241000283984 Rodentia Species 0.000 description 4
- 102000040945 Transcription factor Human genes 0.000 description 4
- 108091023040 Transcription factor Proteins 0.000 description 4
- 238000009825 accumulation Methods 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 210000004204 blood vessel Anatomy 0.000 description 4
- 230000011712 cell development Effects 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- 230000035935 pregnancy Effects 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 101710186200 CCAAT/enhancer-binding protein Proteins 0.000 description 3
- 210000000577 adipose tissue Anatomy 0.000 description 3
- 235000015278 beef Nutrition 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 230000008021 deposition Effects 0.000 description 3
- 235000014113 dietary fatty acids Nutrition 0.000 description 3
- 239000000194 fatty acid Substances 0.000 description 3
- 229930195729 fatty acid Natural products 0.000 description 3
- 150000004665 fatty acids Chemical class 0.000 description 3
- 230000037356 lipid metabolism Effects 0.000 description 3
- 210000001161 mammalian embryo Anatomy 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 102100034808 CCAAT/enhancer-binding protein alpha Human genes 0.000 description 2
- 101710168309 CCAAT/enhancer-binding protein alpha Proteins 0.000 description 2
- 102000008169 Co-Repressor Proteins Human genes 0.000 description 2
- 108010060434 Co-Repressor Proteins Proteins 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 210000000988 bone and bone Anatomy 0.000 description 2
- 230000003139 buffering effect Effects 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 210000002808 connective tissue Anatomy 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 206010020718 hyperplasia Diseases 0.000 description 2
- 229940072221 immunoglobulins Drugs 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 210000001778 pluripotent stem cell Anatomy 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 1
- 102000009122 CCAAT-Enhancer-Binding Proteins Human genes 0.000 description 1
- 108010048401 CCAAT-Enhancer-Binding Proteins Proteins 0.000 description 1
- 102100034798 CCAAT/enhancer-binding protein beta Human genes 0.000 description 1
- 101710134031 CCAAT/enhancer-binding protein beta Proteins 0.000 description 1
- 101100518995 Caenorhabditis elegans pax-3 gene Proteins 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000005698 Frizzled receptors Human genes 0.000 description 1
- 108010045438 Frizzled receptors Proteins 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 102000002254 Glycogen Synthase Kinase 3 Human genes 0.000 description 1
- 108010014905 Glycogen Synthase Kinase 3 Proteins 0.000 description 1
- 108010051975 Glycogen Synthase Kinase 3 beta Proteins 0.000 description 1
- 102100038104 Glycogen synthase kinase-3 beta Human genes 0.000 description 1
- 102000003893 Histone acetyltransferases Human genes 0.000 description 1
- 108090000246 Histone acetyltransferases Proteins 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 206010020880 Hypertrophy Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 101100518997 Mus musculus Pax3 gene Proteins 0.000 description 1
- 101100351033 Mus musculus Pax7 gene Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010028311 Muscle hypertrophy Diseases 0.000 description 1
- 102100038380 Myogenic factor 5 Human genes 0.000 description 1
- 101710099061 Myogenic factor 5 Proteins 0.000 description 1
- 102100032970 Myogenin Human genes 0.000 description 1
- 108010056785 Myogenin Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 108091027981 Response element Proteins 0.000 description 1
- 241000282849 Ruminantia Species 0.000 description 1
- 102000000479 TCF Transcription Factors Human genes 0.000 description 1
- 108010016283 TCF Transcription Factors Proteins 0.000 description 1
- 101001023030 Toxoplasma gondii Myosin-D Proteins 0.000 description 1
- 108010016200 Zinc Finger Protein GLI1 Proteins 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 229960001138 acetylsalicylic acid Drugs 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000003542 behavioural effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000002458 cell surface marker Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 239000000512 collagen gel Substances 0.000 description 1
- -1 compound aspirin compound Chemical class 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 230000032692 embryo implantation Effects 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 239000003630 growth substance Substances 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000008611 intercellular interaction Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 201000010260 leiomyoma Diseases 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000000940 lipogenetic effect Effects 0.000 description 1
- 230000004130 lipolysis Effects 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 210000003141 lower extremity Anatomy 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
- 210000004088 microvessel Anatomy 0.000 description 1
- 230000000394 mitotic effect Effects 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 210000000663 muscle cell Anatomy 0.000 description 1
- 230000004220 muscle function Effects 0.000 description 1
- 230000012042 muscle hypertrophy Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 210000000944 nerve tissue Anatomy 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000003076 paracrine Effects 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000003614 peroxisome proliferator Substances 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 229930002330 retinoic acid Natural products 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 230000022379 skeletal muscle tissue development Effects 0.000 description 1
- 230000004096 skeletal muscle tissue growth Effects 0.000 description 1
- 230000012488 skeletal system development Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000003760 tallow Substances 0.000 description 1
- 230000005758 transcription activity Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
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
- C12N5/0653—Adipocytes; Adipose tissue
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/135—Platelet-derived growth factor [PDGF]
-
- 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
- C12N2501/33—Insulin
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Rheumatology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of methods for purifying the tissue-derived Preadipocyte In Vitro of ox fetal skeletal muscle, the material that the method uses is, 3 monthly age of ox fetus longissimus dorsi muscle tissue, agents useful for same is clostridiopetidase A, fetal calf serum, low sugar Dulbecco modified Eagle medium, cell sorting buffer, antiplatelet source property growth factor receptors Alpha antibodies, dexamethasone, 3-isobutyl-1-methylxanthine, bovine insulin and oil red O.
Description
Technical field
The invention belongs to technological field of biochemistry, and in particular to a kind of tissue-derived preceding body fat of purifying ox fetal skeletal muscle
The method of fat cell.
Background technique
Stem cell is can to maintain duplication and and can be divided into a kind of cell of different type cell ability, stem-cell research
The generation and differentiation regulation that understand cell and tissue are very important.In addition, stem-cell research is for medical domain
Equally there are huge potentiality.As a lot of other organs, skeletal muscle includes many cell types, can generate the satellite of muscle-derived
The fat cell of cell and adipose tissue source property, these are all critically important to Animal husbandry production.It is well known that satellite cell pair
The regeneration of the skeletal muscle of the growth and adult of skeletal muscle is all critically important after birth.Past nearly 50 years thin to isolated satellite
The research of born of the same parents is concentrated mainly on the activation and inhibition of its proliferation, regulates and controls their activity in vitro, raw with other cells such as blood vessel
At intercellular interaction, potentiality between subgroup, and as the potentiality of carrier in gene therapy.In beef cattle production, animal
The growth and development of skeletal muscle and the deposition of intramuscular fat be influence beef production and price main factor.However, at present
Until the research developed for early stage muscle development and intramuscular fat it is relatively fewer, the development of stem cells of especially different cells comes
Source, therefore the stem cell surface molecular marker characteristic for distinguishing different is identified, and establish Differentiation Induction in vitro model
System is of great significance for studying domestic animal muscle and intramuscular fat differentiation.
Embryonic stem cell still has significantly indefinite to the molecular mechanism at orientation flesh, at rouge, at fiber.
However, the embryonic stem cell that quality is proved is separated from a limited number of mammalian species.Moreover, 1981
Embryonic stem cell is separated from inhuman experiment house mouse.Mouse pluripotent stem cell is thoroughly built
It is vertical, by normal growth in embryo transfer to female mice body until birth after being injected into blastaea.In addition, mouse embryonic stem
After the genome of cell can be by transgenosis and homologous recombination technique modification.Genetically engineered cell can be after system genitale be transmitted to
Generation.The human embryonic stem cell line of pluripotency has also been successfully established, and is likewise supplied with the ability for being divided into variety classes cell.To the greatest extent
Pipe to having put into a large amount of effort on agricultural species embryonic stem cell, but be not yet compared with the effort of mouse and the mankind very at
Function.The difficulty of this work may have the following, deposit first is that agricultural species compare the development before embryo implantation with mouse
In the specificity of species, second be growth factor required for agricultural animal embryonic development is not fully understood, third is that
Whether effective stem cell labeling is suitable for agricultural animal and does not know still in mouse.However, the stem cell field of Quick Extended
The research of knowledge, agricultural species aspect embryonic stem cell will also make progress.In fact, the embryonic stem cell of agricultural species develops
There is a unique chance, because they can be used to carry out the test of system genitale, by being injected into involution of embryo in research
It moves on in the parent of replace-conceive, however this test is forbidden in hESC.In addition to this, mouse embryonic stem
Cell should be possible and (have this ability to go to be operated in terms of science of heredity angle), as satellite cell development and functional study
Platform.In the future, if the embryonic stem cell germline from agricultural animal is developed, mouse stem cells research may be just
It can be transferred in the dynamics application study of agricultural species skeletal development.
In mammals, most of structure of skeletal muscles is completed in foetal period growth and development stage.Main muscle fibre is opened
Beginning to be formed is in embryonic period, embryonic phase, and secondary myofibrillogenesis is in people's second trimester of pregnancy and later period, the advanced stage and neonatal period of mouse.Myocyte
Generation is controlled by a series of transcription factors, including Pax3, Pax7, and Gli and four muscle-derived regulatory factor includes MyoD, Myf-5,
Myogenin and MRF-4.Secondary muscle fibre is formed in fat cell, fiber generate lap formation be in people, pig,
Ox, sheep, horse, the second trimester of pregnancy of chicken and rodent latter half of gestation.Muscle-derived, fatty is by embryo with fibroid stem cell
The differentiation of tire stem cell generates.Skeletal muscle stem Cells, which are converted into fatty by muscle-derived, may will increase intramuscular fat deposition.One
Fiber/fat grandmother cell has an impact to intramuscular fat accumulation there may be in skeletal muscle just as fiber in terms of disease.
Muscle cell and fat cell are generated from mesenchymal cell, and mesenchymal cell is present in the getting up early stage of development, especially
It is in the skeletal muscle of foetal period and newborn stage.When most Derived from Mesenchymal Stem Cells becomes myocyte,
Middle sub-fraction difference becomes fat cell --- the basis of intramuscular fat accumulation.Key factor is Wnt family in cell fate
Race's albumen, these albumen are paracrine growth regulators, and there may be different functions in cell development: Wnt signal may
Cause cell Proliferation, apoptosis determines cell fate, variation or precursor maintenance.Classical Wnt access is beta-catenin
Dependence: in conjunction with frizzled protein activate in disorder family protein, family protein has activated glycogen synthase kinase 3, resistance
The beta-catenin for having stopped phosphorylation is then degraded, and the accumulation of beta-catenin is caused.There is no Wnt stimulation, axis albumen/glycogen closes
The antigen degradation of beta-catenin can be improved by the phosphorylation of GSK-3 β at the compound aspirin compound of kinase enzyme 3/.
Stable beta-catenin, which enters after nucleus, deactivates spy with the T cell factor in transcription factor/lymph enhancer effect
Fixed target gene.In the mescenchymal stem cell from marrow, it is raw that the activation of the signal path of Wnt strengthens myocyte
At, it is suppressed that fat generates.Inhibit beta-catenin access that can reduce the total number of myocyte.In Preadipocyte In Vitro
Wnt signal is highly expressed, and inhibits the generation of fat by blocking C/EBP and PPAR γ.Stable beta-catenin at
The Adipose Differentiation of myocyte inhibits related with the fatty generative capacity of the muscle satellite cell increased with the age.
Skeletal muscle stem Cells are present in all skeletal muscle, since position or function are different, can control different hyperplasia
Type/differentiation capability.Postnatal skeletal muscle is influenced extremely sensitive by environment and physiological signal, can be grown up according to required modification
And functional characteristic, for example, movement, injured or damage starts regenerating and repairing for skeletal muscle, although skeletal muscle is by big
Amount is formed by mitotic multicore muscle fibre.Very most is come from present in skeletal muscle to the plasticity of skeletal muscle greatly
Population of stem cells, usually satellite cell.Most of in vitro study is that the back of rodent and the muscle of hind leg divide
Satellite cell from muscle-derived.Nearest one shows satellite cell with the non-Separation Research for ruminating meat animals about ruminating
Specific skeletal muscle separation has been described, but there are shortcoming needs to go to determine, if control is from different muscle
Satellite cell goes to separate.However, a large amount of report display fat cell behavioral difference is depended on by the position where separation cell,
This shows that position may will affect the activity of different tissues cell.For example, different adipose tissue sites have unique growth,
Development and adjusting feature, the enzyme activity of animal dependence.Similar research recently be using purifying culture fat stem cell simultaneously
In cell and the molecule two horizontal researchs carried out.
When needed, satellite cell realizes the function of fusion by starting adult terminal differentiation program.Interesting
It is that we, which constantly discover new growth factor, influences the proliferation of satellite cell, this shows to be possible to additional mechanism and waits being sent out
It is existing.In muscle fibre hypertrophy or repairing, core can be contributed and be merged with existing muscle fibre by satellite cell.Work as flesh
When fiber is for damaging unmoved, satellite cell carries out fusion and forms new myotube finally taking for muscle fibre each other.When
So, skeletal muscle is that a dynamic organization is made of a large amount of elements, including blood vessel, nerve and connective tissue.In Skeletal Muscle Growth and
During regeneration, these elements need to grow and repair, in order to grow need between a thorough functional unit muscle fibre it is mutual
Coordinate.This is supported by previous studies, needs to be related to myocyte's generation, blood vessel to remove to restore muscle function appropriate
Generate the cooperation between nerve formation.Cooperate between myocyte and other cells seemingly possible.Recently, researcher has been
It is presented evidence to support this idea.To describe the interaction between them, researcher has developed common in vitro
The model of culture being made of microvessel fragment and satellite cell.In this system, it is suspended in the separation on collagen gel
MVF is cultivated on the basis of mouse SC monolayer cultivation.In the presence of SC, MVF is illustrated higher compared with MVF is cultivated alone
Angiogenesis index.Satellite is shown more recently by Christov and other people data and endothelial cell is tight in muscle
It is close arranged side by side, it is proposed that directly contact, which may be important, means intercellular exchange.In short, these are preliminary the result shows that one
The satellite cell effect not being found before a is that starting generates program before blood vessel.
However many reports have recorded the external and intrinsic regulation of postnatal muscle-derived satellite cell, plasticity bone
Bone flesh carries out illustration by production capacity and to the response of various cell factor.It is composed according to inflammation and cell factor, bone
Bone flesh will respond in catabolism and anabolism.For example, skeletal muscle is infecting supporter process relevant to existence
It will be decomposed in stage.On the contrary, inflammation eventually results in muscle hypertrophy plus exercise.So far, it has studied in satellite cell
The effect of a variety of type cytokines in activity provide mixing as a result, may be with cell type, dosage is related with the time.
In cellular level, there are two physiological components to exist.One is lipid metabolism, refers into or out fat cell (rouge
Fat generate and lipolysis) energy stream, it does not need stem cell activity.Second is physiology component, referred to as Adipogenesis,
It is the cells switch that can be identified, is shunk by fusiform stem cell, the precursor fatty for being initially formed a shortage lipid is thin
Then born of the same parents form the fat cell of a multiple fat drips, eventually form mature fat cell.In view of what is much delivered every year
About lipid metabolism and lipogenetic article, cannot obtain one substantially effectively reduces internal grease by exogenous processing
Or the method for reducing Adipocyte Differentiation.Really, the article that the major part that field is delivered is delivered is formed in fat cell to think,
Once Preadipocyte In Vitro starts oil accumulation, cell will continue to be carried forward terminal differentiation as participation lipid metabolism
Fat cell.In most animal tallow, the adipocyte number for having oil synthesis and storage capacity does not all have at birth
Explicitly show.Also, adipocytic cell growth is hyperplasia and loose double effects after being born, and the effect of every kind of variation is because of fatty position
It sets and different.It is interesting that should be more fat cells needs in privileged site deposition according to Traditional Thinking, and at fibre
Cell is tieed up in connective tissue portions turn lipoblast.
The peroxidase proliferation receptor (PPAR γ) and CCAAT- enhancer-binding protein of in vitro study display activation
It (C/EBPs) is all the decision sex factor for controlling fat cell and generating.Their induced expression is raw from embryonic stem cell fat
At.Issued a large amount of evidences relevant to agricultural animal support this viewpoint, but slightly not with the mankind and rodent
Same adjustment mechanism.Fat, which generates, originates in foetal period, ruminant second trimester of pregnancy, pig and rodent later period.Adipogenesis
Difference when starting is mainly the difference due to different plant species new born animal in terms of maturity.Adipogenesis is by several passes
The control of key transcription factor, including PPAR γ and C/EBPs.C/EBP β and-δ passes through Adipogenesis stimulant first and is induced, and connects
Getting off is increase by PPAR γ and C/EBP α in terms of expression.C/EBP α and PPAR γ are forced go to open fatty shape each other
It goes to promote Adipogenesis at specific program.The adipogenic main regulation factor of PPAR γ value.PPAR γ is in retinoids x receptor alpha
Heterodimer is formd in target base in the cooperation of (RXR α) and combined peroxisome Proliferators reaction original part
Because in activation.Therefore, vitamin A acid can influence Adipogenesis by the interaction of RXR α and RXR α and PPAR γ.PPAR γ is
One activation ligand of transcription factor.In disabled state, PPAR γ and corepressor contact the transcription activity for suppressing it.Knot
Closing ligand causes corepressor that co-activator is replaced to go to dominate histone acetyltransferase activity such as cAMP response element combination
Albumen ((CBP/p300)).The acetylation of histone causes chromatin division herein and gene to be expressed.Fatty acid is
The receptor of PPAR γ, compared with normal fatty acid, the fatty acid being oxidized seems have higher ability to deactivate PPAR γ.
In short, skeletal muscle tissue development is the central factor of agricultural animal production capacity.Improve skeletal muscle yield and intramuscular
Fat content is the important research content in beef raising production again.Although having carried out largely dry thin about agriculture domestic animal
The research of born of the same parents, but world scientific research personnel is also carrying out extensive work to establish ideal external model at present.In order to divide
From, at flesh/at rouge stem cell, and then the model for being used to establish induction differentiation in vitro is always with purifying skeletal muscle origin
It is also extremely urgent one of work that agricultural animal scientific research personnel carries out for a long time.
Summary of the invention
Aiming at the shortcomings in the prior art, the purpose of the present invention is to provide a kind of purifying skeletal muscle origin fat precursors are thin
The method of born of the same parents.
To achieve the goals above, the technical solution adopted by the present invention is that it is platelet derived by cell surface marker molecule
Growth factor receptors α sorts skeletal muscle origin pluripotent stem cell.
A method of the purifying tissue-derived Preadipocyte In Vitro of ox fetal skeletal muscle, the material that the method uses is,
3 monthly age of ox fetus longissimus dorsi muscle tissue, agents useful for same are that clostridiopetidase A, fetal calf serum, low sugar Dulbecco improve Iger training
Support base, cell sorting buffer, antiplatelet source property growth factor receptors Alpha antibodies, dexamethasone, 3- isobutyl group -1- methyl yellow
Purine, bovine insulin and oil red O.
It the described method comprises the following steps:
Step 1: cell is separately cultured；
Step 2: cell culture；
Step 3: the adipogenic induction of cell；
Step 4: oil red O；
Step 5: oil red O stain test result.
The step 1 specifically includes the following steps:
Step (1) is rinsed several times by ox fetus with 75% alcohol and containing dual anti-phosphate buffer；
Step (2), which is then placed into gnotobasis, carefully breaks amnion, takes out fetus, cuts off back skin with eye scissors
Skin isolates muscle of back with eye scissors, tweezers；
Step (3) is put into culture dish with the musculature that tweezers take out 2 pieces of mung bean grain sizes, is added in advance in culture dish
Enter containing dual anti-phosphate buffer, washes down 3 removing haemocytes with phosphate buffer；
Step (4) is then transferred into EP pipe, and 0.1% type Ⅳ collagenase of 1mL is added, is shredded with eye scissors to meat gruel shape；
The tissue shredded is put into 37 DEG C of gas thermostatic shaking tables by step (5) digests 45min, during which every 15min piping and druming group
It knits primary；
Cytoplasm is filtered with 40 μm of cell filtering net after step (6) 45min, 400rpm is centrifuged 5min, discards supernatant
Cell sorting buffer suspension cell is added in liquid；
Step (7) is incubated for 15min after 2 μ L antiplatelet source property growth factor receptors Alpha antibodies are added, 300rcf centrifugation
10min；
300rcf is centrifuged 10min after 1mL cell sorting buffer is added in step (8), abandons supernatant, is repeated once；
80 μ L cell sorting buffers are added after sucking supernatant in step (9), and it is micro- to add 20 μ L anti-rabbit source immunoglobulins
Pearl is incubated for 15min in 4 DEG C after mixing；
300rcf is centrifuged 10min after 1mL cell sorting buffer is added in step (10), abandons supernatant, is repeated once；
500 μ L cell sorting buffer suspension cells are added in step (11)；
MS sorting column as on magnetic frame, suspension is added on ready MS sorting column, is collected by step (12)
The negative cells flowed down rinse sorting column 3 times every time with 500 μ L cell sorting buffers and collect the liquid flowed down and the first step
Merge；
MS is sorted column and is placed in new collecting pipe from removing on magnetic frame by step (13), and 1mL cell sorting buffering is added
After liquid, quickly it is pushed into piston in sorting column, the liquid for collecting outflow is positive cell.
The step 2 specifically includes the following steps:
When primary cell growth aggregation is to 70~80%, old culture medium is sucked, and clean 2 with phosphate buffer
It is secondary, after 0.25% trypsase is added, it is put into incubator about 30s, it is rounded that cell is observed under inverted phase contrast microscope
When, softly beat culture dish with hand, after circular cells float, at once be added culture medium come terminate digestion reaction continue into
Row, soft piping and druming cell carry out secondary culture with 1:3 ratio, and it is 37 DEG C, CO that cell, which is put into temperature,2The perseverance that content is 5%
It is cultivated in warm incubator.
The step 3 discards culture medium, uses phosphoric acid specifically includes the following steps: when cell growth converges to 100%
Salt buffer cleans 2 times, and lipoblast is added and induces liquid, growth medium adds 0.5 μM of 3-isobutyl-1-methylxanthine, 10
μ g/mL insulin, 1 μM of dexamethasone, start to be induced, every 3d changes a not good liquor.
The step 4 specifically includes the following steps:
Step (a) discards old culture medium, and 4% paraformaldehyde is added in orifice plate, the fixed cell 20min of room temperature；
Step (b) is cleaned 3 times with phosphate buffer；
Step (c) is added under oil red O room temperature and dyes 10min；
Step (d) discards oil red O stain liquid, is cleaned 3 times with phosphate buffer；
Step (e), which is placed under inverted microscope, to be observed the staining conditions of fat drips in cell and takes pictures.
The step 5 specifically includes the following steps:
When cell induces 10d, to Platelet-derived growth factor α-receptor positive cell and platelet derived growth because
Sub- receptor alpha negative cells carry out oil red O stain, the results showed that, the former is more than the latter fat drips, bigger.
Beneficial effect
Mark the fat precursors of the positive group of Platelet-derived growth factor α-receptor screening after purification thin by molecular surface
Born of the same parents form the fat cell with a large amount of fat drips after induction differentiation, and to be formed by fat drips amount seldom for negative group cell.Cause
This, the method is excellent for promoting Preadipocyte purifying to have the effect of.
Detailed description of the invention
The oil red O stain result of cell lipoblast induction differentiation after Fig. 1-2 sorting；
Wherein, Fig. 1 is oil red O stain result after Platelet-derived growth factor α-receptor negative cells break up at rouge；
Fig. 2 is oil red O stain result after Platelet-derived growth factor α-receptor positive cell breaks up at rouge；
Specific embodiment
Embodiment 1
A method of the purifying tissue-derived Preadipocyte In Vitro of ox fetal skeletal muscle, the material that the method uses is,
3 monthly age of ox fetus longissimus dorsi muscle tissue, agents useful for same are that clostridiopetidase A, fetal calf serum, low sugar Dulbecco improve Iger training
Support base, cell sorting buffer, antiplatelet source property growth factor receptors Alpha antibodies, dexamethasone, 3- isobutyl group -1- methyl yellow
Purine, bovine insulin and oil red O.
It the described method comprises the following steps:
Step 1: cell is separately cultured；
Step 2: cell culture；
Step 3: the adipogenic induction of cell；
Step 4: oil red O；
Step 5: oil red O stain test result.
The step 1 specifically includes the following steps:
Step (1) is rinsed several times by ox fetus with 75% alcohol and containing dual anti-phosphate buffer；
Step (2), which is then placed into gnotobasis, carefully breaks amnion, takes out fetus, cuts off back skin with eye scissors
Skin isolates muscle of back with eye scissors, tweezers；
Step (3) is put into culture dish with the musculature that tweezers take out 2 pieces of mung bean grain sizes, is added in advance in culture dish
Enter containing dual anti-phosphate buffer, washes down 3 removing haemocytes with phosphate buffer；
Step (4) is then transferred into EP pipe, and 0.1% type Ⅳ collagenase of 1mL is added, is shredded with eye scissors to meat gruel shape；
The tissue shredded is put into 37 DEG C of gas thermostatic shaking tables by step (5) digests 45min, during which every 15min piping and druming group
It knits primary；
Cytoplasm is filtered with 40 μm of cell filtering net after step (6) 45min, 400rpm is centrifuged 5min, discards supernatant
Cell sorting buffer suspension cell is added in liquid；
Step (7) is incubated for 15min after 2 μ L antiplatelet source property growth factor receptors Alpha antibodies are added, 300rcf centrifugation
10min；
300rcf is centrifuged 10min after 1mL cell sorting buffer is added in step (8), abandons supernatant, is repeated once；
80 μ L cell sorting buffers are added after sucking supernatant in step (9), and it is micro- to add 20 μ L anti-rabbit source immunoglobulins
Pearl is incubated for 15min in 4 DEG C after mixing；
300rcf is centrifuged 10min after 1mL cell sorting buffer is added in step (10), abandons supernatant, is repeated once；
500 μ L cell sorting buffer suspension cells are added in step (11)；
MS sorting column as on magnetic frame, suspension is added on ready MS sorting column, is collected by step (12)
The negative cells flowed down rinse sorting column 3 times every time with 500 μ L cell sorting buffers and collect the liquid flowed down and the first step
Merge；
MS is sorted column and is placed in new collecting pipe from removing on magnetic frame by step (13), and 1mL cell sorting buffering is added
After liquid, quickly it is pushed into piston in sorting column, the liquid for collecting outflow is positive cell.
The step 2 specifically includes the following steps:
When primary cell growth aggregation is to 70~80%, old culture medium is sucked, and clean 2 with phosphate buffer
It is secondary, after 0.25% trypsase is added, it is put into incubator about 30s, it is rounded that cell is observed under inverted phase contrast microscope
When, softly beat culture dish with hand, after circular cells float, at once be added culture medium come terminate digestion reaction continue into
Row, soft piping and druming cell carry out secondary culture with 1:3 ratio, and it is 37 DEG C, CO that cell, which is put into temperature,2The perseverance that content is 5%
It is cultivated in warm incubator.
The step 3 discards culture medium, uses phosphoric acid specifically includes the following steps: when cell growth converges to 100%
Salt buffer cleans 2 times, and lipoblast is added and induces liquid, growth medium adds 0.5 μM of 3-isobutyl-1-methylxanthine, 10
μ g/mL insulin, 1 μM of dexamethasone, start to be induced, every 3d changes a not good liquor.
The step 4 specifically includes the following steps:
Step (a) discards old culture medium, and 4% paraformaldehyde is added in orifice plate, the fixed cell 20min of room temperature；
Step (b) is cleaned 3 times with phosphate buffer；
Step (c) is added under oil red O room temperature and dyes 10min；
Step (d) discards oil red O stain liquid, is cleaned 3 times with phosphate buffer；
Step (e), which is placed under inverted microscope, to be observed the staining conditions of fat drips in cell and takes pictures.
The step 5 specifically includes the following steps:
When cell induces 10d, to Platelet-derived growth factor α-receptor positive cell and platelet derived growth because
Sub- receptor alpha negative cells carry out oil red O stain, the results showed that, the former is more than the latter fat drips, bigger.
Finally, it should be noted that obviously, above-described embodiment is only intended to clearly illustrate the application example, and simultaneously
The non-restriction to embodiment.For those of ordinary skill in the art, it can also do on the basis of the above description
Other various forms of variations or variation out.There is no necessity and possibility to exhaust all the enbodiments.And thus drawn
Shen go out obvious changes or variations still in the protection scope of the application type among.
Claims (1)
1. a kind of method for purifying the tissue-derived Preadipocyte In Vitro of ox fetal skeletal muscle, it is characterised in that: the method uses
Material be 3 monthly age of ox fetus longissimus dorsi muscle tissue, agents useful for same is that clostridiopetidase A, fetal calf serum, low sugar Dulbecco change
Good Eagle's medium, cell sorting buffer, antiplatelet source property growth factor receptors Alpha antibodies, dexamethasone, 3- isobutyl
Base -1- methyl xanthine, bovine insulin and oil red O；
It the described method comprises the following steps: Step 1: cell is separately cultured；Specifically includes the following steps: step (1) is by ox tire
Youngster rinses several times with 75% alcohol and containing dual anti-phosphate buffer；
Step (2), which is then placed into gnotobasis, carefully breaks amnion, takes out fetus, cuts off skin of back with eye scissors, uses
Eye scissors, tweezers isolate muscle of back；
Step (3) is put into culture dish with the musculature that tweezers take out 2 pieces of mung bean grain sizes, is previously added and is contained in culture dish
There is dual anti-phosphate buffer, washes down 3 removing haemocytes with phosphate buffer；
Step (4) is then transferred into EP pipe, and 1mL0.1% type Ⅳ collagenase is added, is shredded with eye scissors to meat gruel shape；
The tissue shredded is put into 37 DEG C of gas thermostatic shaking tables by step (5) digests 45min, and during which every 15min blows and beats tissue one
It is secondary；
Cytoplasm is filtered with 40 μm of cell filtering net after step (6) 45min, 400rpm is centrifuged 5min, discards supernatant liquid, adds
Enter cell sorting buffer suspension cell；
Step (7) is incubated for 15min after 2 μ L antiplatelet source property growth factor receptors Alpha antibodies are added, and 300rcf is centrifuged 10min；
300rcf is centrifuged 10min after 1mL cell sorting buffer is added in step (8), abandons supernatant, is repeated once；
80 μ L cell sorting buffers are added after sucking supernatant in step (9), add 20 μ L anti-rabbit source immunoglobulin microballons, mix
15min is incubated in 4 DEG C after even；
300rcf is centrifuged 10min after 1mL cell sorting buffer is added in step (10), abandons supernatant, is repeated once；
500 μ L cell sorting buffer suspension cells are added in step (11)；
MS sorting column as on magnetic frame, suspension is added on ready MS sorting column, collection flows down by step (12)
Negative cells, rinse sorting column 3 times, use 500 μ L cell sorting buffers every time, collect the liquid that flows down and first step conjunction
And；
MS is sorted column and is placed in new collecting pipe from removing on magnetic frame by step (13), after 1mL cell sorting buffer is added,
It is quickly pushed into piston in sorting column, the liquid for collecting outflow is positive cell；
Step 2: cell culture；Specifically includes the following steps: being sucked when primary cell growth aggregation is to 70~80%
Old culture medium, and cleaned 2 times with phosphate buffer, after 0.25% trypsase is added, it is put into 30s in incubator, is being fallen
Set under phase contrast microscope observe cell it is rounded when, softly beat culture dish with hand, after circular cells float, at once plus
Enter culture medium to continue to terminate digestion reaction, soft piping and druming cell, secondary culture is carried out with 1:3 ratio, cell is put
Enter and is cultivated in the constant incubator that temperature is 37 DEG C, CO2 content is 5%；
Step 3: the adipogenic induction of cell；Specifically includes the following steps: discarding culture when cell growth converges to 100%
Base is cleaned 2 times with phosphate buffer, and lipoblast is added and induces liquid, the lipoblast induction liquid adds for growth medium
0.5 μM of 3-isobutyl-1-methylxanthine, 10 μ g/mL insulin, 1 μM of dexamethasone, start to be induced, every 3d is changed once
Liquid；
Step 4: oil red O；Specifically includes the following steps: step (a) discards old culture medium, 4% paraformaldehyde is added to orifice plate
In, the fixed cell 20min of room temperature；
Step (b) is cleaned 3 times with phosphate buffer；
Step (c) is added under oil red O room temperature and dyes 10min；
Step (d) discards oil red O stain liquid, is cleaned 3 times with phosphate buffer；
Step (e), which is placed under inverted microscope, to be observed the staining conditions of fat drips in cell and takes pictures；
Step 5: oil red O stain test result；Specifically includes the following steps: when cell induces 10d, to platelet derived
Growth factor receptors α positive cell and Platelet-derived growth factor α-receptor negative cells carry out oil red O stain, the results showed that,
The former is more than the latter fat drips, bigger.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610740549.3A CN106282100B (en) | 2016-08-26 | 2016-08-26 | A method of the purifying tissue-derived Preadipocyte In Vitro of ox fetal skeletal muscle |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610740549.3A CN106282100B (en) | 2016-08-26 | 2016-08-26 | A method of the purifying tissue-derived Preadipocyte In Vitro of ox fetal skeletal muscle |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106282100A CN106282100A (en) | 2017-01-04 |
| CN106282100B true CN106282100B (en) | 2019-11-08 |
Family
ID=57677755
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610740549.3A Active CN106282100B (en) | 2016-08-26 | 2016-08-26 | A method of the purifying tissue-derived Preadipocyte In Vitro of ox fetal skeletal muscle |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106282100B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106591224B (en) * | 2017-01-09 | 2020-11-03 | 中国农业科学院北京畜牧兽医研究所 | Method for separating and purifying high-purity chicken precursor intramuscular fat cells and constructing coculture system of high-purity chicken precursor intramuscular fat cells and muscle satellite cells |
| CN106834411B (en) * | 2017-03-24 | 2020-11-10 | 中国农业科学院北京畜牧兽医研究所 | Method for combined analysis of cell proliferation and lipid deposition ability using same adipocyte |
| CN107748130A (en) * | 2017-10-16 | 2018-03-02 | 上海市普陀区中心医院 | A kind of preparation of animal hearts single cell suspension and detection method |
| CN109182260A (en) * | 2018-09-11 | 2019-01-11 | 邵勇 | A kind of method of in vitro culture fetal membrane mescenchymal stem cell |
| CN113355281B (en) * | 2021-06-19 | 2023-04-14 | 浙江大学 | Method for efficiently separating mouse intramuscular fiber-adipogenic progenitor cells |
| CN113717932B (en) * | 2021-09-16 | 2023-03-14 | 四川农业大学 | Primary isolation culture and induced differentiation method for intramuscular precursor adipocytes of adult yaks |
| CN114606182B (en) * | 2022-05-11 | 2022-10-04 | 中国农业科学院北京畜牧兽医研究所 | Passage purification method of sheep embryo-derived myoblasts |
| CN115029305A (en) * | 2022-06-17 | 2022-09-09 | 浙江大学 | Separation and identification method for pig FAPs (FAPs) cells and application of FAPs cells |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060268A (en) * | 2013-01-18 | 2013-04-24 | 青岛农业大学 | Method for culturing goat precursor fat cells in vitro |
-
2016
- 2016-08-26 CN CN201610740549.3A patent/CN106282100B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060268A (en) * | 2013-01-18 | 2013-04-24 | 青岛农业大学 | Method for culturing goat precursor fat cells in vitro |
Non-Patent Citations (2)
| Title |
|---|
| 猪血小板源生长因子受体α基因(PDGFRα) cDNA克隆与表达分析;滕菲等;《农业生物技术学报》;20150731;第23卷(第7期);第931页右栏第1段 * |
| 黑毛和牛肌内前体脂肪细胞分化的基因表达与肌内脂肪性能的关联性研究;陈香凝;《中国优秀硕士学位论文全文数据库》;20131231;第二章第1.4-1.5节，第1.8.1-1.8.2节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106282100A (en) | 2017-01-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106282100B (en) | A method of the purifying tissue-derived Preadipocyte In Vitro of ox fetal skeletal muscle | |
| CN106350480B (en) | A method of the purifying tissue-derived sarcoblast of ox fetal skeletal muscle | |
| CN102329788B (en) | Methods for rejuvenating cells into pluripotent diploid stem cells induced by on nucleus transformation | |
| Dazzi et al. | The role of mesenchymal stem cells in haemopoiesis | |
| CN101748096B (en) | Sub totipotential stem cell and preparation method and application thereof | |
| EP3124600B1 (en) | Method for generating a cell condensate for self-organisation | |
| CN107028981A (en) | Adherent cell from fat or placenta tissue and its purposes in the treatment | |
| US11883438B2 (en) | Sperm activator and uses thereof | |
| US20060247195A1 (en) | Method of altering cell properties by administering rna | |
| CN103930542A (en) | Brown fat cell compositions and methods | |
| JP2007507202A (en) | Cell differentiation of adipose-derived progenitor cells | |
| CN105779383A (en) | Preparation method and application of adipose-derived stem cell-hydrogel three-dimensional cultivation system | |
| Mohammadi-Sangcheshmeh et al. | Isolation, characterization, and mesodermic differentiation of stem cells from adipose tissue of camel (Camelus dromedarius) | |
| CN108779435A (en) | Method for deriving brown fat cell derived from different multipotential stem cells again | |
| CN103215222A (en) | Induction medium for inducing human adipose tissue-derived stromal cells as nerve cells and method | |
| KR101380561B1 (en) | Equine Amniotic Fluid-Derived Multipotent Stem Cells and Method for Producing the Same | |
| CN104152406A (en) | Preparation for enhancing differentiation capacity of chicken skeletal muscle myoblast and application thereof | |
| Medvinsky et al. | Analysis and manipulation of hematopoietic progenitor and stem cells from murine embryonic tissues | |
| CN101506350B (en) | Methods for rejuvenating cells in vitro and in vivo | |
| CN102703380B (en) | Sub-totipotent stem cell, preparation method and application thereof | |
| JP2004129549A (en) | Selective growth method for mesenchymal stem cell from fat-derived cell group | |
| Chaipipat et al. | Primordial germ cells isolated from individual embryos of red junglefowl and indigenous pheasants of Thailand | |
| CN109939128A (en) | A kind of neural stem cell preparation and preparation method thereof | |
| CN102703384B (en) | Application of HPMC (Hydroxy Propyl Methyl Cellulose) hydrogel for impelling adipose-derived stem cells to divide into lymphocytes | |
| Kovpak et al. | The influence of platelet concentrate on the development of cattle embryos in аn in vitro system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |