大鼠骨髓间充质干细胞的分离冻存及向成骨细胞和软骨细胞诱导分化

目的 建立大鼠骨髓间充质干细胞（MSC）体外分离、纯化、增殖、冻存的方法,并观察其体外分化为成骨细胞及软骨细胞的潜能活性.方法 取SD大鼠的股骨及胫骨,冲取骨髓液,采用密度梯度离心法结合贴壁培养法以及单独用贴壁培养法分离纯化MSC,增殖,并测定其生长曲线.MSC在12.5%DMSO条件下经液氮冻存半年复苏,测其存活率.对第3代大鼠MSC进行成骨矿化诱导,测定其碱性磷酸酶（ALP）活性及成骨矿化情况.对第3代大鼠MSC进行诱软骨细胞的诱导分化,测定诱导后的细胞表型情况.结果 大鼠MSC为均一的梭形的成纤维细胞样生长,贴壁及增殖能力强,生长曲线呈S型.MSC于液氮冻存半年后复苏,其存活率为80%.在成骨诱导剂作用下,MSC具有成骨能力,诱导后ALP活性显著提高,并出现矿化结节沉积.在成软骨诱导剂作用下,可见细胞由成纤维细胞样的梭形变为椭圆形及肾形,并分泌异染基质,甲苯胺蓝染色呈阳性,表现为软骨细胞分化特点.结论 获得的SD大鼠MSC生长旺盛,增殖能力强.在特定诱导条件下,大鼠MSC体外可定向分化为成骨细胞及软骨细胞,具有成骨及成软骨分化潜能.

Isolation and cryopreservation of rat bone marrow-derived mesenchymal stem cells and their osteogenic and chondrogenic differentiation potentiality

Objective] To explore the optimal methods of isolating, puring, proliferating,and freezing of SD rat bone marrow-derived mesenchymal stem cells(MSC), and their osteogenic and chondrogenic potentiality. Methods] MSC were harvested from femurs and tibias crest and obtained by gradient centrifuge and adhesive culture methods. Growth curves of passaged MSC were determined. MSC were stored in liquid nitrogen for six months and thawed, then their survival rate was detected by trypan blue staining test. The passaged MSC were cultured in vitro under osteogenic and chondrogenic inductive environments, and their osteogenic and chondrogenic properties were determined. Results] The rat MSC were uniform spindle-shaped in appearance and showed active proliferative capacity and had S shape of growth curve. When MSC were cryopreserved in liquid nitrogen for six months, their viability was 80%.They could be induced to differentiate into osteoblasts under osteogenic induction. Alkaline phosphatase (ALP) activity of the osteogenic inductive cells was higher significantly than that in control group, and these cells could form mineralized nodules. The MSC also could differentiate into chondrocytes by exposure to chondrogenic inductive medium, and the induced cells were positively stained by toluidine blue at various time points. Conclusion] The method of isolating, puring, expanding, and freezing of rat MSC are feasible. The MSC can be cultured, expanded, and cryopreserved stably in vitro. The subcultured MSC have osteogenic and chondrogenic potentiality, so they can be optimal seed cells source for tissue engineering.