HSP27对Hcy诱导的血管内皮细胞损伤的保护作用及其机制研究

目的 研究热休克蛋白27 (HSP27)对同型半胱氨酸(Hcy)诱导的血管内皮细胞损伤的保护作用及其机制.方法 培养人脐静脉内皮细胞,采用MTT法检测Hcy对人脐静脉内皮细胞(HUVECs)存活率的影响;Annexin V-FITC细胞凋亡检测试剂盒及流式细胞术检测Hcy及HSP27对细胞凋亡的影响;设计实验,采用总一氧化氮检测试剂盒(硝酸还原酶法)检测细胞培养液中NO水平、2',7'-二氯荧光黄双乙酸盐(DCFH-DA)活性氧(ROS)检测试剂盒及流式细胞术检测细胞内ROS来研究可能的保护机制.结果 HUVECs与不同浓度的Hcy作用后,0 mmol/L组存活率均值为97.33％,Hcy 0.2 mmol/L组存活率均值为95.17％,Hcy 0.4 mmol/L组存活率均值为90.72％,Hcy 0.6 mmol/L组存活率均值为78.29％,Hcy 0.8 mmol/L组存活率均值为63.65％,Hcy 1.0 mmol/L组存活率均值为41.51％.0 mmol/L组与Hcy 0.2 mmol/L组比较,差异无统计学意义(P＞0.05);Hcy 0.4、0.6、0.8、1.0 mmol/L组存活率均低于0 mmol/L组,差异有统计学意义(均P＜0.05).HSP27浓度越高,细胞的存活率越高,呈浓度依赖性,HSP27对HUVECs损伤具有保护作用.Hcy能够诱发内皮细胞凋亡,而HSP27能显著抑制这种凋亡,表明HSP27能减少Hcy对内皮细胞的损害.HSP27能抑制Hcy引起NO的减少和抑制Hcy诱导ROS的产生.结论 HSP27通过影响NO表达和调节细胞内ROS水平保护Hcy诱导的受损伤内皮细胞.

Protective effect of HSP27 on Hcy induced vascular endothelial cell injury and its mechanism

Objective To study the protective effect of heat shock protein 27(HSP27)on homocysteine(Hcy)induced vascular endothelial cell injury and its mechanism.Methods Human umbilical vein endothelial cells(HUVECs) were cultured.The influence of Hcy on the survival rate of HUVECs was detected by MTT method;the Annexin V FITC apoptosis detection kit and flow cytometry were used to detect the influence of Hcy and HSP27 on cellular apoptosis;the experiment design adopted the total nitric oxide detection kit(nitrate reductase method) for detecting the NO level in cell culture fluid,the DCFH DA reactive oxygen species (ROS) detection kit and flow cytometry were used to detect intracellular ROS to research possible protection mechanism.Results After action of HUVECs with different concentrations of Hcy,the mean values of survival rates in the 0 mmol/L,0.2 mmol/L,0.4 mmol/L,0.6 mmol/L,0.8 mmol/L and 1.0 mmol/L groups were 97.33％,95.17％,90.72％,78.29％,63.65％ and 41.51％ respectively.The difference between the 0 mmol/L group and 0.2 mmol/L group had no statistical significance(P＞0.05);the survival rate in the Hcy 0.4,0.6,0.8,1.0 mmol/L groups was lower than that in the 0 mmol/L group,the difference was statistically significant(P＜0.05).The cellular survival rate was negatively correlated with the Hcy concentration.The higher the HSP27 concentration,the higher the cellular survival rate,showing the concentration dependence.HSP27 had a protective effect on HUVECs damage.Hcy could induce apoptosis of endothelial cells,while HSP27 could significantly inhibit apoptosis,indicating that HSP27 could reduce the damage of Hcy on endothelial cells.HSP27 could inhibit the NO decrease caused by Hcy and inhibited the production of Hcy induced ROS.Conclusion HSP27 protects Hcyinduced injured endothelial cells by influencing NO expression and regulating intracellular ROS level.