TLR4全长及其截断体重组腺病毒对Rf-6A细胞骨架的影响  

EFFECTS OF RECOMBINANT ADENOVIRUS OF FULL-LENGTH AND TRANCATED FORM OF TOLL-LIKE RECEPTOR 4 ON THE ORGANIZATION OF F-ACTIN IN Rf-6A ENDOTHELIAL CELLS

作　　者：宋革[1] 孙学刚[1] 姜勇[1] 

机构地区：[1]第一军医大学病理生理学教研室

出　　处：《生物物理学报》2004年第2期119-124,共6页Acta Biophysica Sinica

基　　金：国家杰出青年科学基金项目(39925014);国家自然科学基金重点项目(39830400);973计划项目(2002CB513000)

摘　　要：为了研究LPS受体TLR4全长及其胞内段缺失的TLR4截断体(驻TLR4)的绿色荧光蛋白重组腺病毒对内皮细胞系Rf-6A骨架蛋白的影响,采用PCR方法扩增目的基因片段,亚克隆至腺病毒穿梭质粒pAdTrack中,用BJ5183细菌同源重组法将目的基因重组于腺病毒骨架载体,重组腺病毒质粒经PacⅠ酶切线性化后,用脂质体法转染293细胞进行腺病毒的包装扩增,用重组腺病毒感染Rf-6A细胞,采用免疫荧光标记方法观察结果。免疫荧光标记结果表明Ad-驻TLR4明显抑制了LPS引起的细胞骨架F-actin的解聚与重排,Ad-TLR4则使LPS引起的F-actin应力纤维产生增强。以上结果说明TLR4全长及其截断体的重组腺病毒感染内皮细胞对LPS诱导的细胞骨架变化具有不同的影响,Ad-驻TLR4对LPS引起的内皮细胞骨架变化具有抑制作用。To investigate the effects of recombinant adenovirus that express full-length and trancated form of toll-like receptor 4(TLR4 and -1TLR4) on the organization of F-actin in cultured endothelial cell line Rf-6A, the sequences of TLR4 and -1TLR4 were amplified by PCR from a TLR4 containing plasmid pcDNA3-TLR4 followed by subcloning of the fragments into a adenovirus shuttle vector pAdTrack to form transfer plasmids, pAdTrack-TLR4 and pAdTrack--1TLR4. After linearization with pme Ⅰ, pAdTrack-TLR4 and pAdTrack--1TLR4 were cotransformed into BJ5183 bacteria that was pretransformed with adenovirus genomic plasmid of pAdEasy-1. The positive recombinants adenovirus plasmid were digested with Pac Ⅰ and transfected into HEK293 cells for the packaging of recombinant adenovirus particles. After infections of the resultant viruses in Rf-6A cells, LPS-induced F-actin depolymerization and reorganization was detected with fluorescence staining with rodamine-phalloidin. The results indicated that LPS stimulation induced F-actin depolymerization and formation of stress fibers. Recombinant adenoviral vectors containing full-length TLR4 intensified LPS-induced F-actin depolymerization, while -1TLR4 recombinant adenovirus inhibited the depolymerization of F-actin induced by LPS in Rf-6A cells significantly. These results revealed that the infection of recombinant adenovirus of full-length and trancated form of TLR4 in cells showed different effects on LPS-induced cellular response. Ad--1TLR4 showed the protection of cultured endothelial cells from injury of LPS-induced depolymerization and reorganization of F-actin.

关 键 词：腺病毒 细胞骨架 TLR4 LPS受体 绿色荧光蛋白 内皮细胞系Rf-6A 骨架蛋白 PCR 脂多糖 

分 类 号：Q78[生物学—分子生物学] R363[医药卫生—病理学]