中华烧伤杂志
中華燒傷雜誌
중화소상잡지
16
2010年
6期
444-447
,共4页
罗敏%梁自文%杨宗城%罗向东
囉敏%樑自文%楊宗城%囉嚮東
라민%량자문%양종성%라향동
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
內皮細胞%顯微鏡檢查,免疫電子%轉染%亞細胞定位%內皮高錶達脂多糖相關因子1%綠色熒光質粒
내피세포%현미경검사,면역전자%전염%아세포정위%내피고표체지다당상관인자1%록색형광질립
Endothelial cells%Microscopy,immunoelectron%Transfection%Subcellular localization%Endothelial overexpressed lipopolysaccharide associated factor 1%Green fluorescent plasmid
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.
目的 瞭解人內皮高錶達脂多糖相關因子1(EOLA1)蛋白在內皮細胞中的亞細胞定位情況.方法 體外培養人臍靜脈內皮細胞株ECV304,另構建增彊型綠色熒光蛋白(EGFP)-EOLA1融閤蛋白錶達質粒.用脂質體分彆將空質粒pEGFP-N2和融閤蛋白錶達質粒EGFP-EOLA1轉染入ECV304細胞.繼續培養48 h,取細胞,用蛋白質印跡法鑒定EGFP及EGFP-EOLA1融閤蛋白的錶達;採用激光共聚焦顯微鏡和免疫電鏡技術,觀察細胞中EOLA1蛋白的亞細胞定位情況.結果 經酶切和測序鑒定證實重組質粒EGFP-EOLA1構建成功.蛋白質印跡法檢測結果顯示,EGFP和EGFPEOLA1融閤蛋白在轉染細胞中成功錶達.激光共聚焦顯微鏡下見轉染空質粒的ECV304,綠色熒光遍佈整箇細胞,但無細胞覈聚集現象;轉染融閤蛋白錶達質粒的ECV304,綠色熒光也呈全細胞分佈,且在胞覈內明顯聚集.透射電鏡下見轉染空質粒的細胞內無免疫沉積物,轉染融閤蛋白錶達質粒的細胞質基質中有免疫沉積物.結論 EOLA1蛋白定位在ECV304細胞覈與細胞質基質中,作為信號轉導因子髮揮作用.
목적 료해인내피고표체지다당상관인자1(EOLA1)단백재내피세포중적아세포정위정황.방법 체외배양인제정맥내피세포주ECV304,령구건증강형록색형광단백(EGFP)-EOLA1융합단백표체질립.용지질체분별장공질립pEGFP-N2화융합단백표체질립EGFP-EOLA1전염입ECV304세포.계속배양48 h,취세포,용단백질인적법감정EGFP급EGFP-EOLA1융합단백적표체;채용격광공취초현미경화면역전경기술,관찰세포중EOLA1단백적아세포정위정황.결과 경매절화측서감정증실중조질립EGFP-EOLA1구건성공.단백질인적법검측결과현시,EGFP화EGFPEOLA1융합단백재전염세포중성공표체.격광공취초현미경하견전염공질립적ECV304,록색형광편포정개세포,단무세포핵취집현상;전염융합단백표체질립적ECV304,록색형광야정전세포분포,차재포핵내명현취집.투사전경하견전염공질립적세포내무면역침적물,전염융합단백표체질립적세포질기질중유면역침적물.결론 EOLA1단백정위재ECV304세포핵여세포질기질중,작위신호전도인자발휘작용.
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.
