低温冻存对兔脂肪间充质干细胞部分生物学特性的影响

Effect of cryopreservation on some biological properties of rabbit adipose-derived mesenchymal stem cells

目的研究低温冻存对兔脂肪间充质干细胞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,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。两组细胞生长曲线呈典型的"S"形,无统计学差异(P>0.05);成脂诱导14 d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(P>0.05)。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。

Objective To study the effect of cryopreservation on some biological properties of rabbit adipose -de-rived mesenchymal stem cells （rADMSCs）.Methods rADMSCs culture was isolated by tissue explants adherent meth-od.Morphology of the primary cells was observed by inverted microscopy .Immunophenotypes of the rADMSCs were deter-mined using flow cytometry .The third passage cells were preserved in liquid nitrogen for 6 months, and then were thawed , and subcultured to passage 7.The growth curves of the cryopreserved cells were analyzed by MTT assay , and the cryopre-served cells were cultured in adipogenic and osteogenic medium , with non-cryopreserved rADMSCs as a control group .The adipogenic and osteogenic abilities of the rADMSCs were evaluated by oil red O staining , alizarin red staining and alkaline phosphatase activity assay , respectively.Results The rADMSCs cultured in vitro exhibited a spindle-shaped appearance and rapid growth expansion .Flow cytometry analysis revealed that the third passage rADMSCs were CD 44-and CD90-posi-tive, but negative for hematopoietic cells surface marker CD 45.The growth curves of cells in the experimental and control groups were “S” shaped, showing a non-significant difference between the two groups （P＆gt;0.05）.The oil red O staining and alizarin red staining results were positive at 2 weeks after adipogenic and osteogenic induction .The ALP activities of the two groups were increased with osteogenic induction time , with a non-significant difference （P＆gt;0.05）.Conclusions Cryopreservation does not affect the growth and differentiation pluripotency of rADMSCs significantly .