骨髓基质细胞的生长特性和转染胰岛素样生长因子-1后的凋亡研究

目的:探讨体外培养大鼠骨髓基质细胞(MSC)的生长特性,同时转染胰岛素样生长因子-1(IGF-1)观察对其凋亡的影响。方法:相差显微镜观察细胞的大体形态,亚甲蓝染色观察细胞集落,细胞计数法研究细胞的增殖能力;逆转录病毒载体pBabe-IGF-1或pBabe转染到MSC,RT-PCR检测IGF-1的表达,流式细胞仪Annexin V-FITC凋亡试剂盒检测细胞凋亡,RT-PCR检测促凋亡基因Bax和抗凋亡基因Bcl-2的表达。结果:骨髓基质细胞主要呈梭形,细胞呈集落性生长,细胞生长到第6代几乎生长停滞;转染IGF-1的MSC组的细胞凋亡率和Bax的表达明显低于其它转染空载体组和对照组,而Bcl-2的表达率则高于后两组(P<0.05)。结论:此方法能较容易地获得大量骨髓基质细胞,IGF-1基因转染后能够明显降低MSC的细胞凋亡,并和Bax基因的升高、Bcl-2基因的降低相关。

Observation of Bone Marrow Stromal Cells' Growth in Vitro and Detection of Their Apoptosis After IGF-1 Transfection

Objective:To investigate the growth characteristics of rat bone marrow stromal cells (MSCs) in vitro and the apoptosis after insulin-like growth factor-1 (IGF-1) gene modification. Methods:The appearance of MSCs was observed under the phase contrast microscope. The cells' ability of colony-formation was determined by staining with methylene blue,and the MSCs growth was quantified by cell counting. The expression of IGF-1 of transfected MSCs was examined with RT-PCR assay,the apoptosis of MSCs after gene modification was observed by flow cytometric analysis and Annexin V-FITC kit. The expression of Bax and Bcl-2 were observed by RT-PCR. Results:Under the phase contrast microscope,the appearance of the MSCs was spindle like and could be expanded rapidly in vitro. MSCs had the ability of colony formation till to the 6th passage. The apoptosis ratio and the expression level of Bax in IGF-1 transfected group were lower than in void transfected group and control group,while the expression level of Bcl-2 was higher (all P<0.05). Conclusion:MSCs can be easily obtained and proliferated in this study,and IGF-1 can decrease the apoptosis ratio,which is associated with the expression of Bax and Bcl-2.