【摘要】 目的研究不同浓度同型半胱氨酸(Hcy)作用不同的时间对人脐静脉内皮细胞中内皮型一氧化氮合酶(eNOS)活性的影响机制。方法取2～3代人脐静脉内皮细胞(HUVECs)与不同浓度Hcy孵育以及孵育不同时间。用Western blot和实时荧光定量RT-PCR法检测热休克蛋白90(HSP90)、蛋白激酶B(Akt)蛋白表达,小凹蛋白(Caveolin-1)、eNOS、P-eNOS蛋白及mRNA的表达,二甲基精氨酸二甲胺水解酶(DDAH)mRNA表达。结果与静息和激活状态下的对照组相比,在Hcy作用下,Caveolin-1蛋白的表达上调(P<0.05),而Akt蛋白的表达下调(P<0.05),且均呈浓度、时间依赖性。eNOS蛋白的表达无明显改变。Hcy对其他检测指标无影响。结论 Hcy引起的eNOS活性降低与增强Caveolin-1蛋白表达使其与eNOS藕联增加,减少HSP90、Akt和eNOS复合物的形成,进而抑制了eNOS的活性有关。更多还原

【Abstract】 Objective To study the mechanism of homocysteine(Hcy) on eNOS activity in human umbilical vein endothelial cells(HUVECs).Methods HUVECs were collected and cultured.Then the second passage of HUVECs were cultured with different Hcy concentrations for different times.The expression of HSP90,Akt,caveolin-1,eNOS,P-eNOS proteins were detected by Western blot and the expression of Caveolin-1,eNOS,DDAH mRNA by RT-PCR.Results Compared with control group,both in base and calcium ionophore activated state,the expression of Caveolin-1 group increased obviously(P<0.05) in concentration and time-dependent manner,but the expression of Akt showed decreasing(P<0.05).The expression of eNOS did not change significantly.There was no difference in the expression of HSP90,eNOS,P-eNOS protein and mRNA among every group.DDAH mRNA among every group too.Conclusions There was no relationship between decreasing activity of eNOS induced by Hcy and eNOS expression,Hcy probably can increase the association of Caveolin-1 and eNOS by enhancing the expression of Caveolin-1,and reduce the complex formation of Caveolin-1,Akt,Caveolin-1 in HUVEC both in base and activated state,which can decrease the activity of eNOS.更多还原