摘要目的 筛选有效抑制血管内皮细胞小窝蛋白-1(Caveolin-1)表达的小干扰RNA(small interferenceRNA,siRNA)序列,并检测转染复合物的细胞毒性. 方法 设计合成针对Caveolin-1的siRNA 3条(siRNA422,siRNA548,siRNA710)及1条带绿色荧光标记的通用阴性对照FAM-siRNA.在siRNAFect介导下转染血管内皮细胞.用实时荧光定量PCR(Real-time PCR)测定转染后血管内皮细胞中Caveolin-1 mRNA的表达,Western blot方法测定干扰后Caveolin-1蛋白表达,比较抑制率,筛选出有效抑制Caveolin-1表达的siRNA.采用磺基罗丹明B法(sulforhodamineB,SRB)分析转染复合物的细胞毒性. 结果 ①在siRNAFect相同剂量下,siRNA 20、30 nmol/L组或40 nmol/L组转染效率均达到85％以上(P＜0.01)；②siRNAFect 1.3μl复合10 nmol/L或20 nmol/L siRNA组细胞存活率均达到80％以上(P＜0.05)；③siRNA548对EA.hy926细胞Caveolin-1基因mRNA抑制效果最明显(P＜0.01)；④与阴性对照组及siRNA422、siRNA710相比较,siRNA548干扰血管内皮细胞48 h后,Caveolin-1蛋白的表达量最低(P＜0.01). 结论 ①siRNA548对血管内皮细胞Caveolin-1表达的抑制效果最大.②siRNA浓度20 nmol/L复合siRNAFect 1.3μl转染效率高,细胞毒性低,适合转染.

abstractsObjective Aim to pick out the small interference RNA (siRNA) which could most effectively inhibit the expression of caveolin-1 in endothelial cells and to detect the cytotoxicity of the transfection complex.Methods Four siRNAs were chemically synthesized:siRNA422,siRNA548,siRNA710 were used to inhibit caveolin-1 expression in endothelial cells,FAM-labeled mismatch siRNA was as a negative control They were all transfected into endothelial cells,respectively.Caveolin-1 mRNA was detected after transfection by real-time PCR and its protein expression was measured by Western blot.The cytotoxicity was analyzed with the method of the sulforhodamine B (SRB).Results ① The transfecfon rates are all above 85％ under the transfection condition of siRNA(20,30 nmol/L or 40 nmol/L) combined with the same dose of siRNAFect(P＜0.01).② The survival rates of transfected cells are all above 80％ under the transfection condition of siRNAFect 1.3 μl combined with siRNA (10 nmol/L or 20 nmol/L)(P＜0.05).③ The expression of caveolin-1 mRNA in EA.hy926 cells was significantly decreased after siRNA548 treatment (P＜0.01).④ Compared with siRNA422,siRNA710,vehicle or mismatch siRNA-treated group,the expression of caveolin-1 protein was the least after siRNA548 treatment 48 h later(P＜0.01).Conclusions ① The siRNA548 can inhibit caveolin-1 expression most effectively in endothelial cells.② siRNA(20 nmol/L) combined with siRNAFect(1.3 μl) has lower cytotoxicity and higher transfection efficiency,which is suitable for transfection.