鼠骨髓间充质干细胞对缺氧/复氧心肌细胞的影响

Effect of bone marrow mesenchymal stem cells on hypoxia/reoxygenation cardiac myocytes in rats

目的:观察在体外培养条件下骨髓间充质干细胞(mesenchymal stem cell,MSC)对缺氧/复氧新生大鼠心肌细胞的保护作用。方法:无菌条件下分离、培养、传代Wistar大鼠MSC,细胞融合达90%时更换培养基培养24h,收集细胞培养液作为MSC条件培养基;原代培养新生Wistar大鼠心肌细胞,先用5%CO2、10%H2、85%N2混合气造成细胞缺氧24h,再给予95%O2和5%CO2混合气复氧2h,建立缺氧/复氧模型。MSC处理组于复氧时加入MSC条件培养基,正常细胞作为对照组,分别于复氧后用MTT法、乳酸脱氢酶(lactated dehydrogenase)法测定各组心肌细胞活性的改变;RT-PCR观察细胞凋亡蛋白Bcl-2和Bax的表达。结果:与对照组比较,缺氧/复氧组细胞活力显著降低(P<0.01)。与缺氧/复氧组比较,MSC处理组细胞活力明显增加(P<0.01)。缺氧/复氧组抗凋亡基因Bcl-2的表达明显降低,促凋亡基因Bax的表达明显增加,而MSC条件培养基可以减少Bax基因的表达,提高Bcl-2/Bax值,与缺氧/复氧组比较差异具有显著性(P<0.01)。结论:MSC对体外缺氧-复氧诱导的心肌细胞的凋亡有保护作用。

Objective To observe the protective effect of mesenchymal stem cell （MSC） on newly born rat＇s cardiac muscle cells under hypoxia/reoxygenation injury in vitro. Methods MSCs of Wistar rat was sterilizedly isolated, cultured and passaged. When cells fusion reached 90%, the medium was replaced and MSC was further cultured 24 h. The supernatant of cell culture was harvested as MSC condition medium. Primary culture of Wistar rat＇s cardiac myocytes was performed in vitro. Hypoxia/reoxygenation model of cardiac myocytes was established. The methods were as follows ： gas mixture of 5% CO2, 10% H2 and 85% N2 resulted in hypoxia of cells for 24 h, and then cells were cultured in the condition of gas mixture of 95% 02 and 5% CO2 for 2 h. MSC condition medium was added to MSC group in the process of reoxygenation. MSC condition medium wasn＇t added to pure hypoxia/reoxygenation group in the process of reoxygenation. Normal cells were as control group. Activities of cardiac myocytes were analyzed by MTT and leakage rate of lactated dehydrogenase （LDH） respectively. Expression of anti-apoptosis Bcl-2 gene and promoting apoptosis Bax gene were detected by RT-PCR. Results Viability of cardiac myocytes in pure hypoxia/reoxygenation group significantly decreased, compared with control group （P 〈 0.01 ）. Viability of cardiac myocytes in MSC group significantly increased （P 〈 0.01 ）, compared with pure hypoxia/reoxygenation group. Expression of Bcl-2 gene significantly decreased and Bax gene significantly incresasd in pure hypoxia/reoxygenation group, while MSC could reduce expression of Bax gene and improve Bel-2/Bax ratio. Morever, pure hypoxia/reoxygenation group and MSC group had obviously difference （P 〈 0.01）. Conclusion MSC could protect apoptosis of hypoxia/reoxygenation cardiac myocytes in vitro.