磷酸胆碱聚合物载AT1受体siRNA对血管内皮细胞AT1受体表达的影响

Effect of phosphorylcholine polymer MPC30-DEA70 carried siRNA on AT1 receptor expression in vascular endothelial cells

目的探讨新型阳离子磷酸胆碱聚合物MPC30-DEA70载AT1受体siRNA对血管内皮细胞AT1受体表达的影响。方法应用琼脂糖凝胶电泳表征具有不同N/P比值的MPC30-DEA70与siRNA的复合物;将不同比例的MPC30-DEA70/siRNA基因复合物转染至人脐静脉内皮细胞,应用荧光显微镜(FM)检测其细胞内分布;流式细胞术(FCM)检测转染效率及荧光强度;Western blot法和RT-PCR法检测AT1受体蛋白及mRNA的表达情况。结果不同N/P比值的基因复合物在电泳中可见不同程度的迟滞现象,随正电性增强而显著。FM观察到MPC30-DEA70/siRNA复合物分布在细胞核周围,FCM结果显示随N/P比值的增大,转染效率明显增加,荧光强度也随之增强。RT-PCR法和Western blot法显示随着N/P比值的增大,基因复合物可使AT1受体的mRNA和蛋白表达明显下降(P<0.05)。结论 MPC30-DEA70可作为AT1受体siRNA的载体转染至血管内皮细胞,下调AT1受体的mRNA及蛋白的表达,提示阳离子磷酸胆碱聚合物可以有效负载和运输siRNA,抑制特异基因的表达,是一种有效的非病毒类转基因载体。

Objective To investigate the effect of cationic phosphorylcholine polymer MPC30-DEA70 carried siRNA on AT1 receptor expression in vascular endothelial cells.Methods The MPC30-DEA70/siRNA complexeswith different N/P ratioswere formed and transferred into human umbilical vein endothelial cell line（HUVEC）.Inverted fluorecent microscopy（FM） was used to detect the intracellular distribution of the complexes.The transfection efficiency and fluorescent intensity were detected by flow cytometry（FCM）.The expression of AT1 receptor proteins and mRNA were detected by western blot and RT-PCR.Results The MPC30-DEA70/siRNA complexes with different N/P ratios shows different degrees of electrophoretic retardation,this phenomenon accordance with the enhancement of positive electric.Results came from FM present that MPC30-DEA70/siRNA complexes mainly located around the cell nucleus.FCM analysis showed that the transfection efficiency and fluorescent intensity of the complexes significantly enhanced as the increase of N/P ratios.RT-PCR and Western blot showedthat the expression of AT1 receptor mRNA and protein in endothelial cells were significantly reduced as the the increase of N/P ratios.Conclusions The MPC30-DEA70 can be used as the carrier of the AT1 receptor siRNA into the vascular endothelial cells,MPC30-DEA70/siRNA complexes decreased the expression of AT1 receptor mRNA and protein.It indicates that the cationic phosphorylcholine polymer can transfer siRNA into the vascular endothelial cells effectively,inhibit expressionof specific gene,and it might be a kind of effective non-virus gene carrier.