低温冻存对兔脂肪间充质干细胞部分生物学特性的影响的研究.DOC

低温冻存对兔脂肪间充质干细胞部分生物学特性的影响的实验研究 刘琴，陈芳，杨丽君，王丽平，朱以良，张宜 （解放军广州军区武汉总医院医学实验科，湖北省武汉市430070） 【摘要】目的 研究低温冻存后对兔脂肪间充质干细胞的部分生物学特性的影响。方法 采用组织块法分离培养兔脂肪间充质干细胞。用倒置显微镜观察原代细胞的细胞形态，流式细胞仪检测兔脂肪间充质干细胞的免疫表型。取第3代兔脂肪间充质干细胞置于-196℃液氮保存半年，37℃复苏并传至第7代。实验分为两组，实验组为冻存复苏后传至第7代的兔脂肪间充质干细胞，对照组为未冻存的第7代兔脂肪间充质干细胞，取复苏后传至第7代的兔脂肪间充质干细胞，用MTT绘制其生长曲线；添加成脂、成骨诱导液进行诱导，油红O、茜素红染色和碱性磷酸酶活性检测分别进行鉴定。结果 体外培养的兔脂肪间充质干细胞呈梭形纤维样细胞形态，生长力旺盛。流式细胞仪检测显示，第3代兔脂肪间充质干细胞强表达CD44、CD90，阴性表达造血细胞相关的表面标志CD45。冻存复苏后传至第7代的兔脂肪间充质干细胞生长力旺盛，两组细胞生长曲线呈典型的“S”形，无统计学差异（P0.05）；成脂诱导14d后，油红O染色呈阳性；成骨诱导2周时茜素红染色阳性，ALP表达活性随成骨诱导时间延长不断增加且无统计学差异（P0.05）（P0.01），于成骨诱导13d达平台期。结论 冻存后的兔脂肪间充质干细胞体外生长及多向分化潜能未发生显著变化。 【关键词】间充质干细胞；免疫表型；成脂诱导；成骨分化；低温冻存 【中图分类号】[中图分类号] R394.2 【文献标识码】[文献标识码] A Effect of Cryopreservation cryopreservation on partial partial Bbiological Features features of Rabbit rabbit adipose-derived mesenchymal stem cells Liu Qin, Chen Fang, Yang Li-jun, Wang Li-ping, Zhu Yi-liang, Zhang Yi （Department of Medical Laboratory, Wuhan General Hospital of Guangzhou Military Command, Wuhan Hubei 430070, China） 【Abstract】[Abstract] Objective To study the effect of cryopreservation on partial biological features of rabbit adipose-derived mesenchymal stem cells(rADMSCs). Methods rADMSCs were isolated by tissue explants adherence method. Morphology of primitive cells was observed by inverted microscopy. Immunophenotypes of rADMSCs were measured using flow cytometry. The third generation cells were stored with a simple method of cryopreservation by slow cooling with dimethyl sulphoxide as a cryoprotectant and rapid thawing. After cryopreserved in liquid nitrogen for 6 months, cells were thawed, and subcultured to passage 7. The grow curve of the cryopreserved seventh passage cells were analyzed by MTT. , and the cryopreserved cells were cultured in adipogenic and osteogenic medium, with non-cryopreserved rADMSCs as the control group. Upon induction with adipogenic and osteogenic inductive medium, tThe adipogenic and osteogenic abilitiesability of passage