靶向心肌细胞TLR4基因小干扰RNA载体构建  

Construction and Efficacy Evaluation of the siRNA Expression Vector Targeting the TLR4 Gene of Cardiomyocytes

作　　者：郑勇萍[1] 李梦云[1] 柯剑娟[1] 吴云[1] 何祥虎[1] 张宗泽[1] 王焱林[1] 

机构地区：[1]武汉大学中南医院麻醉科,湖北武汉430071

出　　处：《武汉大学学报（医学版）》2016年第6期929-933,共5页Medical Journal of Wuhan University

基　　金：国家自然科学基金资助项目(编号:81471858)

摘　　要：目的:设计合成有效靶向Toll样受体4(TLR4)基因小干扰RNA(siRNA)表达载体,且挑选TLR4基因稳定沉默心肌细胞。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-U6neo质粒连接、酶切、测序并鉴定。脂质体法转染心肌细胞,新霉素(G418)加压筛选并收集稳定表达质粒的心肌细胞,并观察光镜下心肌细胞形态。RT-PCR及Western Blot法检测收集的心肌细胞TLR4mRNA和蛋白的表达,确定siRNA对TLR4的抑制效率。结果:测序鉴定插入发夹样序列正确,成功构建了TLR4基因siRNA表达载体;并获得了稳定沉默TLR4的心肌细胞。未见细胞毒性形态学改变。多种方法均证实siRNA作用于TLR4基因后,心肌细胞TLR4 mRNA和蛋白表达均被抑制,其中pSilence2.1-siTLR4-1抑制作用最强。结论:成功构建了靶向TLR4基因siRNA表达载体,并有效抑制心肌细胞TLR4表达。Objective：To construct vectors expressing small interfering RNA targeting the Toll like receptor-4（TLR4）gene and obtain cardiomyocytes with TLR4 knocked down.Methods：Three small hairpin RNAs（shRNAs）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 cardiomyocytes by the cationic lipid methods respectively.Cardiomyocytes were stably transfected with an expression plasmid and screened by G418.Cardiomyocytes were observed under optical microscope.TLR4 mRNA and protein expression were detected by RT-PCR and Western Blot methods respectively.Results：The pSilence2.1-siTLR4 expression vectors were successfully constructed and a TLR4 knockeddown cardiomyocytes line was established.No cytotoxic morphological changes were observed in cardiomyocytes.RT-PCR and Western Blot analysis confirmed that the expression of TLR4 was significantly down-regulated in the infected cardiomyocytes,and pSilence2.1-siTLR4-1 was the most efficacious recombinant vector.Conclusion：Recombinant vectors carrying shRNA targetingthe TLR4 gene were successfully constructed and the TLR4 expression in cardiomyocytes was inhibited.

关 键 词：TOLL样受体4 RNA干扰 RNA 小分子干扰 心肌细胞 

分 类 号：R54[医药卫生—心血管疾病]