兔羊膜间充质干细胞的体外分离培养及鉴定

Isolation,Culture and Identification of Rabbit Amniotic Mesenchymal Stem Cells in Vitro

为寻找兽医领域新的干细胞来源,以兔羊膜组织为材料,使用胰蛋白酶对羊膜组织进行消化去除上皮细胞,再使用Ⅰ型胶原酶消化分离培养得到兔羊膜间充干细胞(rabbit amniotic mesenchymal stem cells, rAMSCs)。观察细胞形态和生长规律并绘制出生长良好的P3及P10代rAMSCs生长曲线。经对比发现,P3代生长优于P10代,所以选择P3代进行冻存,探索P3代rAMSCs的冻存及复苏能力。通过免疫荧光鉴定相关干细胞标志蛋白的表达;通过流式细胞术对rAMSCs的细胞表型进行鉴定;通过体外诱导分化鉴定rAMSCs的诱导分化潜能。结果表明:P3代rAMSCs形态为长梭形,增殖性能好,冻存后复苏率高。P3代rAMSCs能够成功表达干细胞标志蛋白OCT4、SOX2和NANOG,低表达细胞表型蛋白CD34、CD45和HLA-DR,高表达细胞表型蛋白CD29、CD49d和CD73,符合MSCs鉴定标准,并在体外诱导条件下能够分化为成脂细胞和成骨细胞。说明酶消化法能够成功获取rAMSCs,并且生物学特性稳定,表达干细胞相关蛋白及表型,具有诱导分化潜能。

Looking for new source of stem cells in the veterinary field to rabbit amniotic membrane used as material,the use of trypsin to digest remove epithelial cells,amniotic membrane organization type reuseⅠcollagenase digestion separation between cultured rabbit amniotic fill stem cells(rabbit amniotic mesenchymal stem cells,rAMSCs).To observe the cell morphology and growth rule and map out good P3 and P10 generation rAMSCs growth curve.By contrast,we found that P3 grow better than P10 generation,so we choose P3 generation for frozen storage,and explore the P3 generation rAMSCs frozen storage and recovery ability.Immunofluorescence was used to identify the expression of related stem cell marker proteins.The cell phenotypes of rAMSCs were identified by flow cytometry.The induced differentiation potential of rAMSCs was identified through induction differentiation in vitro.The results showed that the P3 rAMSCs were long spindle shaped,with good proliferation performance and high recovery rate after cryopreserve.RAMSCs in the P3 generation were able to successfully express stem cell marker proteins OCT4,SOX2 and NANOG,low expression of cell phenotypes CD34,CD45 and HLA-DR,and high expression of cell phenotypes CD29,CD49 d and CD73,which met the MSCs identification criteria,and were able to differentiate into lipid-generating cells and osteoblasts under in vitro induction conditions.This experiment showed that the enzyme digestion method could successfully obtain rAMSCs with stable biological characteristics,expression of stem cell-related proteins and phenotypes,and induced differentiation potential.