目的：建立稳定的体外细胞缺氧模型，探讨人体脐静脉内皮细胞（HUVEC）在缺氧条件下RhoA蛋白和Rho激酶对其内皮细胞内皮型一氧化氮合酶（eNOS）表达的影响。方法：利用jetPEI-HUVEC、siRNA分别转染SH-SY5Y细胞、HEK293细胞和HUVEC后制备体外细胞缺氧模型，通过细胞裂解对相关蛋白进行免疫印迹分析。结果：常氧条件下RhoA蛋白在HUVEC中表达水平较低，但在缺氧条件下培育5 h后表达增加，同时缺氧3 h后Rho激酶表达增加，5 h后达高峰，而eNOS的表达恰恰相反。缺氧条件下，活化型RhoA蛋白下调eNOS的表达，而siRNA使RhoA蛋白表达减少从而上调eNOS的表达；Rho结合域抑制Rho激酶活性而上调eNOS的表达。结论：RhoA蛋白和Rho激酶的表达及活化抑制内皮细胞中eNOS的表达，因此可以通过某些药物如他汀类或Rho激酶抑制剂，抑制RhoA蛋白和Rho激酶的活性，从而增加eNOS的表达水平，对心脑血管疾病产生保护作用。%Objective: To establish stable hypoxic model in vitro and to explore the effect of RhoA protein/Rho-kinase in human umbilical vein endothelial cell, (HUVEC) on eNOS expression of its endothelial cell under hypoxic condition.Methods: SH-SY5Y cells, HEK293 cells and HUVEC were respectively transfected using jet-PEI-HUVEC and siRNA to prepare cellular hypoxic model, the Western blotting analysis was preformed for the related proteins through cellular lysis.Results: RhoA protein levels in HUVEC were low under normoxic conditions, but were signiifcantly increased after 5 h of hypoxia. Endothelial Rho-kinase expression was not de-tected until 3 h of hypoxia; such expression remained signiifcantly increased after 5 h. On the other hand, endo-thelial eNOS expression was similar after 3 h of hypoxia, but was signiifcantly decreased after 5 h. The hypoxia-induced decrease in eNOS expression was signiifcantly enhanced by expression of the constitutively active form of RhoA and signiifcantly inhibited by suppression of RhoA expression by small interfering RNA. The hypoxia-induced decrease in eNOS expression was signiifcantly inhibited when endogenous Rho-kinase activation was inhibited by Rho-binding domain expression.Conclusion: Expression and activation of RhoA and Rho-kinase inhibit eNOS expression in endothelial cells, attempts to down-regulate RhoA and Rho-kinase by multiple drugs, such as statins or Rho-kinase inhibitors, might provide endothelial and cardiovascular beneifts through upregula-tion of eNOS.
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