RNA干扰抑制内皮细胞韦伯潘力氏小体的释放

RNA interference inhibits Weibel-Palade body release from endothelial cells

背景:利用RNA干扰和沉默基因达到临床治疗效果,已经越来越受到重视。目的:探讨利用小干扰RNA方法抑制内皮细胞韦伯潘力氏小体释放的效果和意义。方法:设计腺病毒介导的针对调节韦伯潘力氏小体释放的关键蛋白N-己基顺丁烯二酰亚胺敏感因子N端功能区shRNA,筛选鉴定收获病毒,转染人主动脉内皮细胞,携带N-己基顺丁烯二酰亚胺敏感因子shRNA的腺病毒感染设为实验组、单纯病毒表达载体转染设为阴性对照组,空白对照组未加任何东西。结果与结论:用携带N-己基顺丁烯二酰亚胺敏感因子shRNA的腺病毒感染内皮细胞后,3组N-己基顺丁烯二酰亚胺敏感因子mRNA表达比较,差异有显著性意义(P<0.05);实验组的表达随时间变化持续下降,24,48,72h组间比较差异有显著性意义(P=0.048)。N-己基顺丁烯二酰亚胺敏感因子蛋白表达实验组低于两对照组,差异有显著性意义(P<0.05);而两对照组之间比较差异无显著性意义(P=0.249)。免疫荧光染色显示,N-己基顺丁烯二酰亚胺敏感因子shRNA腺病毒感染,明显抑制凝血酶诱导的韦伯潘力氏小体的释放。说明携带N-己基顺丁烯二酰亚胺敏感因子shRNA的腺病毒感染人主动脉内皮细胞,能明显抑制N-己基顺丁烯二酰亚胺敏感因子mRNA及蛋白表达,抑制凝血酶诱导的韦伯潘力氏小体释放。

BACKGROUND： RNA interference and silent genes have got more and more attention as they can achieve the clinical effect.OBJECTIVE： To explore the effect of small hairpin RNA in inhibiting the Weibel-Palade body release in endothelial cell.METHODS： A small hairpin RNA mediated with an adenovirus vector was designed depending upon the N-terminal functional area of N-ethylmaleimide sensitive factor which act as a key protein in Weibel-Palade body release in endothelial cell. Being screened and identified, the harvested viruses were transfected into human aortic endothelial cell. The adenovirus vector with N-ethylmaleimide sensitive factor small hairpin RNA was regarded as the experimental group, and the pure adenovirus vector was regarded as negative control group, the blank control group without any treatment.RESULTS AND CONCLUSION： There was significant difference of N-ethylmaleimide sensitive factor mRNA expression in the endothelial cell in three groups after transfected with N-ethylmaleimide sensitive factor small hairpin RNA （P 〈 0.05）; the expression of N-ethylmaleimide sensitive factor mRNA in the experimental group was decreased with time prolonged, and there was significant difference of the N-ethylmaleimide sensitive factor mRNA expression in the experimental group at 24, 48 and 72 hours （P=0.048）. The expression of N-ethylmaleimide sensitive factor protein in the experimental group was lower than that in the negative control group and blank control group, and the difference was significant （P 〈 0.05）; there was no significant difference between negative control group and blank control group （P=0.249）. The immunofluorescence staining showed that the release of Weibel-Palade body in endothelial cell induced with thrombin was inhibited obviously after transfected with N-ethylmaleimide sensitive factor small hairpin RNA. The expression of N-ethylmaleimide sensitive factor mRNA and protein in endothelial cell transfected with harvested adenoviruses which carried the N-ethylmaleimide sensitive factor small hairpin RNA were significantly decreased, and the release of Weibel-Palade Body induced with thrombin was inhibited dramatically.