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目的探讨骨髓间充质干细胞(MSC)对缺氧诱导心肌细胞凋亡的保护作用及可能机制。方法分离培养成年大鼠 MSC 和乳鼠心肌细胞,将培养的乳鼠心肌细胞进行缺氧处理后,加入MSC 或其条件培养液继续在无氧(95%N_2+5%CO_2,持续缺氧组)或正常氧(缺氧/复氧组)条件下共培养72 h,采用 Hoechst 细胞核染色,计算凋亡细胞百分率;采用蛋白质免疫印迹法检测凋亡相关蛋白 Bcl-2和 Bax 的变化。结果持续缺氧可以诱导心肌细胞凋亡,凋亡率为51.6%±2.4%,与 MSC或其条件培养液共培养,心肌细胞凋亡率显著减低,分别为15.1%±5.4%和24.0%±4.2%(均 P<0.01);缺氧/复氧损伤可以引起心肌细胞凋亡,凋亡率为20.9%±2.7%,心肌细胞与 MSC 共培养,凋亡率显著减低(11.5%±3.7%,P<0.05);心肌细胞与 MSC 条件培养液共培养,凋亡率略有减低(20.1%±4.2%,P>0.05)。蛋白质免疫印迹显示凋亡时心肌表达 Bax 水平较高,与 MSC 或其条件培养液共培养时,Bax 表达水平呈不同程度降低,与心肌细胞凋亡发生率减低一致,Bcl-2无明显变化。结论 MSC 对体外缺氧诱导的心肌细胞的凋亡有保护作用,其作用机制可能是通过细胞间直接接触和旁分泌细胞因子,影响了 Bcl-2家族部分蛋白分子在心肌细胞的表达。
Objective To investigate the protective effect of bone marrow mesenchymal stem cells (MSCs) on hypoxia-induced cardiomyocyte apoptosis and its possible mechanism. Methods The adult rat MSC and neonatal rat cardiomyocytes were isolated and cultured. The cultured neonatal rat cardiomyocytes were treated with hypoxia, then added with MSC or its conditioned medium to continue in hypoxia (95% N 2 + 5% CO 2, continuous hypoxia group ) Or normal oxygen (hypoxia / reoxygenation group) for 72 h. The percentage of apoptotic cells was calculated by Hoechst staining. The expressions of apoptosis-related proteins Bcl-2 and Bax were detected by Western blotting. Results Continuous hypoxia induced cardiomyocyte apoptosis with a apoptotic rate of 51.6% ± 2.4%. Co-culture with MSC or its conditioned medium significantly reduced the rate of cardiomyocyte apoptosis, which were 15.1% ± 5.4% and 24.0% ± 4.2% (all P <0.01). Hypoxia / reoxygenation injury induced apoptosis of cardiomyocytes with a apoptotic rate of 20.9% ± 2.7%. Co-culture of cardiomyocytes and MSCs resulted in a significant decrease of apoptosis rate (11.5% ± 3.7% , P <0.05). The rate of apoptosis decreased slightly (20.1% ± 4.2%, P> 0.05) after co-cultured cardiomyocytes with MSC conditioned medium. Western blotting showed that the level of Bax in myocardium was higher during apoptosis, and the expression of Bax was decreased to some extent when co-cultured with MSC or its conditioned medium, which was consistent with the decrease of cardiomyocyte apoptosis rate, but no change of Bcl-2. Conclusion MSC could protect cardiomyocytes from apoptosis induced by hypoxia in vitro. The mechanism may be through the direct contact between cells and the secretion of cytokines, which may affect the expression of some Bcl-2 protein in cardiomyocytes.