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目的观察黄芪甲苷预处理对无血清及缺氧诱导的骨髓间充质干细胞(MSCs)凋亡的影响,并探讨其作用机制。方法采用密度梯度离心法分离培养大鼠MSCs,分别以40,80,160μg·m L-1低、中、高剂量的黄芪甲苷预处理MSCs 24 h,然后进行无血清及缺氧培养24 h,RT-PCR法检测细胞中Bcl-2、Bax、Caspase-3的基因表达,Western-blot法检测细胞中Bcl-2、Bax以及Caspase-3的蛋白表达。结果密度梯度离心法可有效分离大鼠MSCs;无血清及缺氧诱导可上调Bax以及Caspase-3基因及蛋白的表达,下调Bcl-2基因及蛋白表达;黄芪甲苷可下调Bax及Caspase-3基因及蛋白的表达,上调Bcl-2基因及蛋白表达。结论黄芪甲苷可抑制无血清及缺氧诱导的MSCs凋亡,其作用机制可能是通过上调抗凋亡基因,下调促凋亡基因,由此减少促凋亡蛋白的释放,阻止Caspase级联反应的启动。
Objective To observe the effect of astragaloside preconditioning on apoptosis of bone marrow mesenchymal stem cells (MSCs) induced by serum-free and hypoxia and to explore its mechanism. Methods Rat MSCs were isolated and cultured by density gradient centrifugation. MSCs were pretreated with low, medium and high dose of Astragaloside 40, 80, 160μg · m L-1 for 24 h respectively, then cultured for 24 h in serum- The gene expression of Bcl-2, Bax and Caspase-3 were detected by RT-PCR and the protein expression of Bcl-2, Bax and Caspase-3 were detected by Western-blot. Results Density gradient centrifugation could effectively separate rat MSCs; serum-free and hypoxia-induced up-regulation of Bax and Caspase-3 gene and protein expression and down-regulation of Bcl-2 gene and protein expression; Astragaloside IV downregulation of Bax and Caspase-3 Gene and protein expression, up-regulate Bcl-2 gene and protein expression. Conclusion Astragaloside IV can inhibit the apoptosis of MSCs induced by serum and hypoxia. The mechanism may be that by up-regulating the anti-apoptotic genes and down-regulating the pro-apoptotic genes, it can reduce the release of pro-apoptotic proteins and prevent Caspase cascade The start