Objective To study the effect of siRNA on inhibition of Weibel-Paiade body(WPB) release in endothelial cells in order to prevent cardiovascular disease and develop siRNA drugs. Methods A small hairpin RNA(shRNA) , which regulates the adenovirus-mediated WPB releasing key protein,a N-ethylmaleimide sensitive factor(NSF) in the N-terminal functional area,was designed. The harvested viruses were identified and transfected into human aortic endothelial cells (HAEC). Inhibitory effect of shRNA on expression of NSF mRNA and protein was detected by RT-PCR and Western blot,respectively. Effect of shRNA on WPB release in endothelial cells was observed with immunofluorescence staining. Results The expression level of NSF mRNA and protein in endothelial cells transfected with adenoviruses was significantly lower in experimental group than in control group 24,48 and 72 h after transfection(P = 0. 048). However,no significant difference was found in expression of NSF protein between experimental group and control group (P>0. 05). Immunofluorescence staining showed that adenovirus-transfected HAEC significantly inhibited the thrombin-mediated WPB release. Conclusion Adenovirus-transfected HAEC can significantly inhibit the expression of NSF mRNA and protein and WPB release, which may provide certtain reference for the prevention and treatment of atherosclerosis and acute coronary syndrome.%目的 探讨利用RNA干扰方法抑制内皮细胞韦伯潘力氏小体(WPB)释放的效果和意义,为防治心血管病和开发小分子RNA药物奠定基础.方法 设计腺病毒介导的针对调节WPB释放的关键蛋白N-乙基顺丁烯二酰亚胺敏感因子(NSF)N端功能区的小发卡RNA(shRNA),筛选鉴定收获病毒,使用NSF-shRNA转染人主动脉内皮细胞为实验组、阴性对照病毒感染为阴性组、不加任何干扰为空白组,RT-PCR和Western blot法观察对NSFmRNA及蛋白表达的抑制作用,免疫荧光染色观察对WPB释放的影响.结果 用携带NSF shRNA的腺病毒感染内皮细胞后,实验组NSF mRNA表达与空白组(P=0.02)及阴性组(P=0.035)比较,差异有统计学意义；实验组NSF mRNA表达随时间延长持续下降,24、48及72 h明显下降,差异有统计学意义(P=0.048).实验组NSF蛋白表达,与空白组(P=0.031)及阴性组(P=0.004)比较,差异有统计学意义；而空白组与阴性组差异无统计学意义(P=0.249).免疫荧光染色显示,NSF-shRNA腺病毒感染,明显抑制凝血酶诱导的WPB释放.结论 携带NSF-shRNA的腺病毒感染人主动脉内皮细胞,能明显抑制NSF mRNA及蛋白表达,抑制凝血酶诱导的WPB释放,对未来动脉粥样硬化及急性冠状动脉综合征的防治有一定的参考价值.
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