siRNA沉默热休克蛋白27对雌激素心血管保护作用的影响

Influence of siRNA-mediated HSP27 knockdown on estrogen-mediated cardiovascular protection

摘要:

目的  通过基因沉默技术，研究热休克蛋白27（HSP27）对雌激素介导的心血管保护作用的影响。方法  人脐静脉内皮细胞（HUVEC）转染HSP27 siRNA，免疫荧光法检测siRNA转染效率，Western blot和逆转录-聚合酶链反应（RT-PCR）检测HUVEC中HSP27蛋白和mRNA表达水平；1×10-7 mol/L雌二醇（E2）处理转染HSP27 siRNA的HUVEC，24 h后采用硝酸还原酶法检测培养基中一氧化氮(NO)的含量，酶联免疫吸附法（ELISA）检测细胞间黏附分子-1（ICAM-1）、内皮素-1（ET-1）的含量。结果  HSP27 siRNA能降低HSP27蛋白和mRNA表达。1×10-7 mol/L E2可促进HUVEC分泌NO，同时降低ICAM-1和ET-1水平。转染HSP27 siRNA后，E2介导内皮细胞分泌NO和ICAM-1受到抑制。但沉默HSP27对E2抑制内皮细胞分泌ET-1无明显影响。结论  HSP27可能通过雌激素来影响内皮细胞分泌NO和ICAM-1，参与心血管的保护。

Abstract:

Objective To investigate the influence of HSP27 on the cardiovascular protection of estrogen by gene silencing technique. Methods HSP27 siRNA was transfected to human umbilical vein endothelial cells (HUVECs). The transfection efficiency was assessed by immunofluorescence technique. The expressions of HSP27 mRNA and protein in HUVECs were determined by RT-PCR and Western blot, respectively. HUVECs transfected with HSP27 siRNA were incubated with 1×10-7 mol/L estradiol (E2) for 24 hours. The level of nitric oxide (NO) in medium was determined by nitrite/nitrate colorimetric method, and the levels of intercellular adhesion molecule-1 (ICAM-1) and endothelin-1 (ET-1) in the supernatant were determined by ELISA. Results HSP27 siRNA sequence significantly reduced the expression of HSP27 in protein and mRNA levels. E2 at the level of 1×10-7 mol/L could obviously promote the secretion of NO from HUVECs, and decrease the ICAM-1 and ET-1 levels. After transfection with HSP27 siRNA, E2-mediated secretion of NO and ICAM-1 by HUVECs was inhibited. However, the level of ET-1 had no significant change. Conclusions HSP27 may influence the role of E2 on the secretion of NO and ICAM-1 in HUVECs, and protect the cardiovascular system.