野生型与Pro370Leu突变型myocilin基因真核细胞表达的对比研究  

Comparative study of eukaryotic cell expression of wild-type and Pro370Leu mutation type myocilin gene

作　　者：王建文[1] 孙慧敏[1] 季建[1] 李筱荣[1] 郑建秋[1] 

机构地区：[1]天津医科大学眼科中心,300070

出　　处：《中华医学杂志》2006年第10期700-704,共5页National Medical Journal of China

摘　　要：目的比较Pro370Leu突变型myocilin(mMYOC)与野生型myocilin(wMYOC)基因在真核细胞中表达的差别,了解原发性开角型青光眼(POAG)的发病机制。方法将携带有wMYOC基因的绿色荧光蛋白重组表达载体pEGFPN3wMYOC和携带有mMYOC基因的红色荧光蛋白重组表达载体pDsRed2N1mMYOC,分别及共同转染真核细胞Hela,用荧光显微镜观察蛋白表达定位情况,并用Western印迹分析蛋白分泌情况。结果荧光显微镜观察到wMYOC蛋白和mMYOC蛋白均成功表达,都定位在细胞质中,但wMYOC蛋白分布均匀,而mMYOC蛋白呈聚集状态。共表达时,wMYOC蛋白也呈聚集状态,且与mMYOC蛋白有共定位倾向。经Western印迹分析,相应的转染细胞可分泌wMYOC蛋白,而不能分泌mMYOC蛋白;当共表达时,也没有wMYOC蛋白分泌。结论两种基因都能成功表达,都定位在细胞质;mMYOC蛋白表现为聚集倾向和分泌障碍,并且影响wMYOC蛋白的表达和分泌特性。Objective To compare the eukaryotic cell expression of wild-type myocilin （MYOC） （wMYOC） gene and Pro370Leu mutation type MYOC（mMYOC） gene so as to understand the mechanism of primary open angle glaucoma （POAG）. Methods HeLa ceils were cultured and then transfected with the vector pEGFP-N3-wMYOC, recombinant plasmid with wild type MYOC gene and enhanced green fluorescein gene, or the vector pDsRed2-N1-mMYOC, recombinant plasmid with mutation MYOC gene and red fluorescein gene, respectively, or co-transfected with these 2 plasmids. Corresponding blank vectors pEGFPN3 and pDsRed2-N1 were used as markers. Fluorescence microscopy was used to observe the localization of red fluorescence and green fluorescence. Laser co-focusing microscopy was used to observe the effect of co-transfection. The green and red fluorescein antibodies were examined by Western blotting. Results Green fluorescence was observed in the cytoplasm of the HeLa ceils transfected with the blank vector pEGFP-N3, in an even distribution; and red fluorescence was observed in the cytoplasm of the HeLa cells transfected with the blank vector pDsRed2-N1, in an even distribution too. The ceils transfected with pEGFP-N3-wMYOC showed evenly-distributed green fluorescence in the cytoplasm, and the ceils transfected with pDsRed2-N1- mMYOC showed red fluorescence in the cytoplasm in a state of aggregation. Both red and green fluorescence could be seen in the cells co-transfected with pEGFP-N3-wMYOC and pDsRed2-N1-mMYOC, both in a state of aggregation and co-localization. Laser co-focusing microscopy showed the same results. Protein with the relative molecular weight of 83 000, identical to that of the recombinant protein of wMYOC protein and green fluorescein, could be found in the culture fluid and ceil lysate of the pEGFP-N3-wMYOC-transfected cells; however, could be found in the lysate only but not in the culture fluid of the co-transfected ceils. Protein with the relative molecular weight of 81 000, identical to that of the recombinant prote

关 键 词：青光眼 转染 质粒 基因 MYOC 

分 类 号：R346[医药卫生—基础医学]