大鼠骨髓间充质干细胞的分离、鉴定与冻存及对细胞活性的影响

Separation,Identification and Cryopreservation of Bone Marrow Mesenchymal Stem Cells in Rats and Their Effects on Cytoactive

目的分离培养大鼠骨髓间充质干细胞(MSCs),观察冻存对细胞活性的影响,为其临床应用奠定基础。方法取3周龄SD雄性大鼠,断颈处死,取股骨和胫骨,提取骨髓MSCs进行体外培养,流式细胞仪鉴定细胞表面的阳性标记物和阴性标记物。所得骨髓MSCs-70℃冻存,分别于冻存前、冻存后1周、1个月、6个月取细胞,采用MTT法绘制骨髓MSCs增殖曲线,采用诱导实验检测骨髓MSCs的成骨细胞与脂肪细胞分化的能力。结果分离大鼠骨髓MSCs表面标志物CD29阳性表达率为97.9%,CD90阳性表达率为97.4%。冻存后1周、1个月和6个月,大鼠骨髓MSCs复苏率差异无统计学意义(P>0.05)。冻存、冻存时间延长对细胞增殖活力及多分化能力,经条件诱导后成钙结节数目和油滴数目组间差异无统计学意义(P>0.05)。结论本实验成功提取了大鼠骨髓MSCs,配置的冻存体系也可较长时间保持MSCs活性,为其后继研究打下了基础。

Objective To separate and culture bone marrow mesenchymal stem cells（ MSCs）,and to observe effect of cryopreservation on cytoactive so as to provide foundation for its clinical application. Methods SD male rats with 3 weeks old were selected and killed by decollation,and thighbone and tibia were used to extract MSCs for culture in vitro. Positive and negative markers on MSCs surfaces were detected by flow cytometry. All selected MSCs were stored under-70℃ circumstance,and before and in 1 week,1 month and 6 months after cryopreservation the cells were revived,and then MTT assay was used to draw cumulative growth curve,and differentiated capacities of osteoblast and adipocyte were detected by induced assay. Results The positive rates of MSCs superficial positive markers CD29 and CD90 were 97. 9% and 97. 4% respectively,and reanimation rates of MSCs in 1 week,1 month and 6 months after cryopreservation were no significant differences among groups（ P〉0. 05）. There were no significant differences in effects of cryopreservation time and prolonging cryopreservation time on cell multiplication activity and multi-differentiation ability,numbers of calcium nodus and oil drop after condition induction between groups（ P〉0. 05）. Conclusion In this experiment,bone marrow MSCs in rats are extracted successfully,and the frozen storage system may maintain MSCs activity for a long time,which provides a foundation for subsequent study.