骨髓间充质干细胞对氧诱导心肌细胞凋亡的保护作用

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％N2＋5％CO2,持续缺氧组）或正常氧（缺氧／复氧组）条件下共培养72h,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。

Anti-apoptotic effects of mesenchymal stem cells on cardiac myocytes: in vitro study with rats

OBJECTIVE: To investigate the anti-apoptotic effects of mesenchymal stem cells (MSCs) on hypoxia-injured cardiac myocytes. METHODS: MSCs were isolated from the bone marrow of 6 - 8 week-old Sprague-Dawley rats. Cardiac myocytes from neonatal rat were cultured under hypoxia, then the hypoxia-injured cardiac myocytes were divided into 3 groups: cultured alone (control group), co-cultured with the MSCs, or co-cultured in MSC-conditioned media in conditions of anoxia (95% N(2) + 5% CO(2), continuous hypoxia group) or normoxia [hypoxia/reoxygen (H/R) group] for 72 hours. The cell apoptosis was measured by Hoechst staining, and Western blotting was used to test the protein expression of Bcl-2 and Bax in the cardiac myocytes. RESULTS: The apoptotic rate of the cardiac myocytes cultured under hypoxia was 51.6% +/- 2.4%, significantly higher than those of the cardiac myocytes co-cultured with MSCs and MSC-conditioned media respectively (15.1% +/- 5.4% and 24.0% +/- 4.2% respectively, both P < 0.001). The apoptotic rate of the H/R group was 20.9% +/- 2.7%, significant higher than that of the MSC group (11.5% +/- 3.7%, P < 0.05), however, not significantly different from that of the MSC-conditioned media group (20.1% +/- 4.2%, P > 0.05). The protein expression of Bcl-2 was not significantly different among different groups. The Bax protein expression of the MSC group and MSC-conditioned media group were 2.28 +/- 0.46 and 3.01 +/- 0.26 respectively, both significantly lower than that of the control group (7.62 +/- 1.28, both P < 0.05). The decreased expression of Bax in the cardiac myocytes was greatly related to the decreasing of apoptosis. CONCLUSION: Co-cultured MSCs show significant anti-apoptotic effects on cardiac myocytes both in continuous hypoxia and in H/R conditions with the possible mechanism of direct cell to cell interaction and paracrine of cytokines which effect the expression of Bax in the myocytes.