人内皮高表达脂多糖相关因子1蛋白亚细胞定位研究  

Subcellular localization of human endothelial-overexpressed lipopolysaccharide-associated factor 1 protein

作　　者：罗敏[1] 梁自文[1] 杨宗城 罗向东 

机构地区：[1]第三军医大学西南医院内分泌科,重庆400038 [2]全军烧伤研究所,创伤、烧伤与复合伤国家重点实验室

出　　处：《中华烧伤杂志》2010年第6期444-447,共4页Chinese Journal of Burns

基　　金：国家自然科学基金（30300101、30371468、30670838）

摘　　要：目的 了解人内皮高表达脂多糖相关因子1（EOLA1）蛋白在内皮细胞中的亚细胞定位情况.方法 体外培养人脐静脉内皮细胞株ECV304,另构建增强型绿色荧光蛋白（EGFP）-EOLA1融合蛋白表达质粒.用脂质体分别将空质粒pEGFP-N2和融合蛋白表达质粒EGFP-EOLA1转染入ECV304细胞.继续培养48 h,取细胞,用蛋白质印迹法鉴定EGFP及EGFP-EOLA1融合蛋白的表达;采用激光共聚焦显微镜和免疫电镜技术,观察细胞中EOLA1蛋白的亚细胞定位情况.结果 经酶切和测序鉴定证实重组质粒EGFP-EOLA1构建成功.蛋白质印迹法检测结果显示,EGFP和EGFPEOLA1融合蛋白在转染细胞中成功表达.激光共聚焦显微镜下见转染空质粒的ECV304,绿色荧光遍布整个细胞,但无细胞核聚集现象;转染融合蛋白表达质粒的ECV304,绿色荧光也呈全细胞分布,且在胞核内明显聚集.透射电镜下见转染空质粒的细胞内无免疫沉积物,转染融合蛋白表达质粒的细胞质基质中有免疫沉积物.结论 EOLA1蛋白定位在ECV304细胞核与细胞质基质中,作为信号转导因子发挥作用.Objective To study the subcellular localization of human endothelial-overexpressed lipopolysaccharide-associated factor 1 （ EOLA1 ） protein in endothelial cells. Methods Human umbilical vein endothelial cell strain ECV304 were cultured in vitro. The fusion protein of enhanced green fluorescent protein （EGFP）-EOLA1 expressing plasmid was constructed. Empty plasmid with EGFP at N side （pEGFPN2 ） and fusion protein expressing plasmid EGFP-EOLA1 was respectively transfected into ECV304 cells with liposome. After being cultured for 48 hours, the expression levels of EGFP and fusion protein EGFP-EOLA1 in cells were detected with Western blot. The subcellular localization of EOLA1 protein was detected by laser scanning confocal microscope and immunoelectron microscopy. Results The EGFP-EOLA1 coexpression plasmid was verified to be successfully constructed by enzyme cutting and gene sequencing. The fusion protein of EGFP-EOLA1 was observed to express in transfected cells through Western blot. Green fluorescence scattered all over the ECV304 cells transfected with empty plasmid and cells transfected with fusion protein expressing plasmid, and it gathered obviously in the nuclei in the latter cells. Immune deposits were observed in the matrix of cells transfected with fusion protein expressing plasmid but not in the cells transfected with empty plasmid. Conclusions EOLA1 protein is localized in the nucleus and the matrix of ECV304 cell, and it plays its role as a signal transduction factor.

关 键 词：内皮细胞 显微镜检查 免疫电子 转染 亚细胞定位 内皮高表达脂多糖相 关因子1 绿色荧光质粒 

分 类 号：R686[医药卫生—骨科学]