机械拉伸刺激对大鼠骨髓间充质干细胞增殖的调节

Regulation for proliferation of rat bone marrow mesenchymal stem cells by mechanical stretch

目的以大鼠骨髓间充质干细胞(rat marrow mesenchymal stem cells,rMSCs)为实验对象,考察拉伸的加载对体外的调节作用。方法采用密度梯度离心法分离培养rMSCs,免疫细胞化学染色观察细胞表面相关抗原的表达,流式细胞技术分析细胞周期分布。采用拉伸加载实验装置对培养细胞进行不同应变大小和作用时间的拉伸加载,MTT分析rMSCs的增殖,RT-PCR技术检测c-fos基因表达变化。结果Percoll密度梯度离心结合差异贴壁法能有效分离rMSCs,在含15%胎牛血清的DMEM培养基中稳定生长,呈长梭形或纺锤形。rMSCs均一表达CD44和Fibronectin,不表达CD34,G_0/G_1期细胞占90%以上。拉伸刺激后rMSCs的OD值增加,在研究的时间(15～60 min)和应变范围(2%～8%)内表现出时间和应变依赖特征。RT-PCR结果表明rMSCs在1．0Hz、8%应变条件下拉伸60 min后,c-fos基因表达明显增加。结论研究提示,rMSCs的体外生长受机械拉伸作用的调节:适宜的拉伸刺激可明显促进体外培养rMSCs的增殖。

Objective To investigate the regulation of proliferation of rat marrow mesenchymal stem cells （rMSCs） by mechanical stretch. Methods rMSCs were isolated and purified by density gradient＇centrifugation, expression of relative surface antigens was detected by immunochemistry staining, and distribution of cell cycle was analyzed with flow cytometry, rMSCs were exposed to stretch with different time and magnitude, MTT （3-（4,5-dimethylthiazol-2-yl）-2, 5-diphenyl tetrasodium bromide） was used for proliferation assay and expression of c-fos gene was examined by RT- PCR. Results rMSCs could be perfectly achieved by the combination of density gradient centrifugation and adhesion difference, and cultured stably in DMEM medium containing 15% fetal bovine serum （FBS）. The cells exhibited an elongated fusiform shape and CD44 and Fibronectin positive while CD34 negative. Flow cytometry revealed that G0/G1 phase cells amount to 90%. MTT assay showed that O.D values of rMSCs increased after exposure to stretch, and revealed a time- and strain-dependence manner within all time and stretch ranges studied. In contrast to control group, expression of c-fos gene in rMSCs showed a significantly increase after exposure to 1Hz, 8% strain for 60 minutes. Concluslon mechanical stimulation is crucial to regulating the cell growth and proliferation. The appropriate mechanical stretch could be an accelerant for proliferating capacity of rMSCs in vitro.