RNA干扰技术建立细丝蛋白表达下调的血管平滑肌细胞模型

Establishment of filamin downregulated VSMC model using RNAi technigue

目的利用RNA干扰(RNAi)技术使细丝蛋白(FLN)mRNA表达降低,抑制FLN在血管平滑肌细胞(VSMC)中的生成。方法采用RNAi技术建立FLN表达下调VSMC模型,设计3条可诱发FLN mRNA表达基因沉默的小分子RNA(SiRNA)片段,根据构建质粒载体并转染VSMC的不同,建立7-1、7-2、7-3组和正常对照组、空白载体组5组转染细胞模型,荧光显微镜下观察绿色荧光蛋白标记物的表达并鉴定转染效率,荧光定量PCR鉴定FLN mRNA抑制效果。Western blot检测FLN蛋白的表达。结果设计的3条SiRNA均可有效抑制FLN mRNA的表达。其中7-1和7-2组抑制效果最明显,可达60%以上。3组FLN/肌动蛋白(actin)的比值与正常对照组或空白载体组相比较均明显下降。结论利用RNAi抑制FLN在VSMC中的生成,有可能减少病理状态下收缩表型VSMC向合成表型转化。有可能达到从基因水平上预防或治疗动脉粥样硬化的作用。

Objective To reduce the expression of filamin（FLN）mRNA in VSMC by RNAi technigue in order to inhibit the expression of FLN protein in VSMC,which may interrupt the switch of VSMC from contractile phenotype to synthetic phenotype under the pathologic condition. Methods Three fragments of SiRNA were designed,which could induce decrease in FLN mRNA expression, then the plasmid was constructed to transfect the rabbit VSMC and 7-1,7-2, 7-3 groups corresponding to the 3 fragments were established. The green fluorescent protein（GFP） in VSMC was observed to assess the efficacy of transfection by fluorescence microscopy. The expression of FLNmRNA and FLN was assessed with fluorescent quantitation reahime PCR and Western blot respectively. Results All the three SiRNA could inhibit the expression of FLNmRNA. The expression of FLN in target groups were decreased by 30% to 50% and the most significant effect was seen in 7-1 and 7-2 groups. Conclusion The expression of target gene can be sup pressed effectively by RNA interference. When the FI.NmRNA in VSMC was suppressed, the switch from contractile phenotype to synthetic phenotype may be redued in pathologic state, which may play a role in prevention or treatment of atherosclerosis at gene level.