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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。
OBJECTIVE: To investigate the growth characteristics of rat bone marrow stromal cells (MSCs) cultured in vitro and to observe the effect of insulin-like growth factor-1 (IGF-1) on its apoptosis. Methods: The morphological changes of cells were observed by phase contrast microscopy, cell colonies were observed by methylene blue staining and cell proliferation was evaluated by cell counting method. The retroviral vector pBabe-IGF-1 or pBabe was transfected into MSC and IGF-1 The apoptosis was detected by flow cytometry Annexin V-FITC kit, and the expression of Bax and anti-apoptotic gene Bcl-2 were detected by RT-PCR. Results: The bone marrow stromal cells were spindle-shaped with colony-forming cells. The cells grew to the 6th generation and almost stopped growing. The apoptosis rate and the expression of Bax in MSC-1 cells transfected with IGF-1 were significantly lower than those in other transfected cells Vector group and control group, while the expression of Bcl-2 was higher than the latter two groups (P <0.05). Conclusion: This method can easily obtain a large number of bone marrow stromal cells. IGF-1 gene transfection can significantly reduce the apoptosis of MSCs, and is related to the increase of Bax gene and the decrease of Bcl-2 gene.