缺氧预适应诱导人脐静脉内皮细胞释放的微囊泡对正常H9c2细胞的作用

Effects of hypoxia preconditioning induced human umbilical vein endothelial cells microvesicles on normal H9c2 cells

目的:分离提取缺氧预适应(HPC)诱导人脐静脉内皮细胞(HUVECs)分泌的微囊泡(MVs),探讨HPC-EMVs对正常H9c2心肌细胞的作用和其Caspase 3、Bcl-2和Bax表达的影响。方法:采用HPC方法诱导HUVECs释放MVs,将其和正常培养的大鼠H9c2心肌细胞孵育,通过MTT法检测细胞存活率和比色法测定细胞培养液中乳酸脱氢酶(LDH)活性;通过Hoechst 33258染色观察细胞核的形态变化;比色法测定培养液中Caspase 3活性以及Western blot法检测H9c2细胞中Bcl-2和Bax的表达。结果:HPC模型构建过程中,与缺氧/复氧(H/R)组相比,HPC组细胞存活率显著升高(P<0.01),即成功构建HUVECs的HPC模型;HPC-EMVs处理H9c2细胞结果发现,与对照组H9c2细胞相比,HPC-EMVs 30、60μg/m L组细胞存活率显著降低(P<0.001),且LDH活性和Caspase 3活性显著升高(P<0.01或P<0.001),凋亡细胞量增加,Bcl-2/Bax比值显著降低(P<0.001)。结论:成功采用HPC的方法诱导HUVECs释放MVs,HPC-EMVs通过影响正常H9c2细胞Caspase 3、Bcl-2和Bax蛋白的表达发挥促凋亡作用。

Objective:To investigate the effects of microvesiclesc(MVs)on normal H9c2cardiomyocytes and the expression of Caspase3,Bcl-2and Bax.Methods:MVs were generated from HUVECs induced by hypoxia solution pH6.8/5.8and pre-hypoxia10min/reoxygenation15min,which were incubated with normal H9c2cells.Cell viability was tested by MTT assay.The activity of lactate dehydrogenase(LDH)in cell culture medium was tested by colorimetric method.Morphological changes of H9c2cells were observed by Hoechst33258staining.Caspase3activity was determined by colorimetry and the expression of Bcl-2and Bax in H9c2cells were detected by Western blot.Results:HUVECs HPC model was successfully established.Compared with H/R group,the cells viability of HPC group was significantly increased(P<0.01).Compared with normal H9c2cells,the cells viability of HPC-EMVs30,60μg/mL groups were significantly decreased(P<0.01),and the release of LDH increased(P<0.001),and the ratio of Bcl-2/Bax significantly decreased(P<0.001).Conclusion:HUVECs HPC model is successfully established,and MVs are generated from HUVECs induced by the method of HPC,and HPC-EMVs have pro-apoptotic effect on normal H9c2cells by affecting the expression of Caspase3,Bcl-2and Bax.