靶向血管平滑肌细胞TLR4基因小干扰RNA载体构建及效果评价

Construction and Efficacy Evaluation of siRNA Expression Vector Targeting TLR4 Gene of Vascular Smooth Muscle Cells

目的设计合成有效的靶向Toll样受体4(TLR4)基因的小干扰RNA(siRNA)表达载体,并筛选出TLR4基因稳定沉默的血管平滑肌细胞(SMC)。方法依据RNA干扰(RNAi)序列设计原则,以TLR4基因为靶基因,合成3对小发夹RNA(shRNA)寡核苷酸链,经退火、与线性化pSilence 2.1-U6 neo质粒连接,酶切及测序进行鉴定。脂质体法转染血管SMC,新霉素(G418)加压筛选并收集稳定表达质粒的SMC。RT-PCR及Western blot法检测收集的SMC中TLR4 mRNA和蛋白表达,以确定siRNA对TLR4的抑制效率。结果测序鉴定插入的发夹样序列正确,成功构建了TLR4基因siRNA表达载体;并获得了稳定沉默TLR4的血管SMC。RT-PCR及Western blot证实RNA干扰TLR4基因后,SMC中TLR4 mRNA和蛋白表达均明显减低,其中pSilence2.1-siTLR4-1的抑制作用最强。结论成功构建了靶向TLR4基因的siRNA表达载体,并有效抑制血管SMC中TLR4表达。

Objective To construct vectors expressing small interfering RNA targeting the Toll like receptor-4 （TLR4） gene and obtain TLR4 knock downed vascular smooth muscle cells （SMC）. Methods Three small hairpin RNA （shRNA） targeting the TLR4 gene were designed, synthesized and cloned into the pSilence 2.1-U6 neo vector. Positive clones were verified with double enzyme digestion and sequencing. Then the recombinants were transfected to SMC by the cationic lipid method respectively.SMC were stably transfected with an expression plasmid and screened by G418. TLR4 mRNA and protein expression were detected by RT-PCR and Western blot methods. Results The pSilence2.1-siTLR4 ex-pression vectors were successfully constructed and a TLR4 knock-downed SMC cell line was established. RT-PCR and Western blot analysis confirmed that the expression of TLR4 was significantly down-regulated in the infected SMC cell line, and pSilence2.1-siTLR4-1was the most efficacious recombinant vector.Conclusion Recombinant vectors carrying shRNA targeting the TLR4 gene were successfully constructed and the TLR4 expression in vascular SMCs was inhibited.