人转化生长因子β诱导基因真核表达载体的构建及其对人角膜上皮细胞增生的影响  

Construction of eukaryotic plasmid expressing human transforming growth factor beta-induced gene and its influence on human corneal epithelial cell

作　　者：牛静宜[1] 刘婧[1] 李晓霞[2] 陈建苏[2] 徐锦堂[2] 钟敬祥[1] 

机构地区：[1]暨南大学附属第一医院眼科,广州510630 [2]暨南大学医学院眼科实验室,广州510632

出　　处：《中华实验眼科杂志》2011年第12期1071-1076,共6页Chinese Journal Of Experimental Ophthalmology

基　　金：广东省自然科学基金项目（10151063201000044）

摘　　要：背景人转化生长因子-β诱导（TGFBI）基因是第一个被确定的角膜营养不良的致病基因，而TGFBI引起角膜营养不良的机制目前尚不清楚，研究TGFBI的功能对于揭示角膜营养不良的发病机制及了解角膜生理及病理功能都具有重要意义。目的构建人TGFBI基因的真核表达载体并将其转染于人角膜上皮细胞，探讨其对角膜上皮细胞增生及相关基因表达的影响。方法从角膜移植后剩余的供体角膜中提取人正常角膜组织总RNA，经逆转录聚合酶链反应（RT—PCR）合成TGFBI cDNA，将TGFBI胶回收产物与载体pCMV—N-HA分别进行双酶切，取连接产物10μl转化100μl大肠杆菌感受态DH5α克隆入真核表达载体pCMV—N—HA，以菌落PCR和EcoRV、XhoI双酶切法测序鉴定。设置重组质粒pCMV—N—HA—TGFBI转染组、空质粒pCMV—N—HA转染组、阴性对照组及pGFP—C2转染对照组，以测定重组质粒转染率。重组质粒pCMV-N—HA—TGFBI转染人角膜上皮细胞，激光共焦显微镜下观察转染pGFP—C2后增强型绿色荧光蛋白（EGFP）的表达，用细胞计数试剂盒8（CCK-8法）检测转染细胞的增生情况，SYBR荧光实时定量PCR和Western blot法检测TGFBI、基质金属蛋白酶（MMPs）、组织金属蛋白酶抑制剂（TIMP）蛋白及其mRNA在转染后角膜上皮细胞的表达。结果pCMV—N-HA—TGFBI阳性克隆质粒进行经EcoRV和XhoI双酶切鉴定测序结果显示，扩增的TGFBI cDNA以正确序列和方式插入载体，pGFP—C2载体转染人角膜上皮细胞后48h共焦显微镜下可见EGFP的表达，转染效率为70％。重组质粒pCMV—N—HA-TGFBI转染组TGFBI mRNA的表达明显高于空质粒pCMV—N—HA转染组和阴性对照组，TGFBI蛋白仅在pCMV—N-HA．TGFBI转染组表达。CCK-8法显示重组质粒pCMV—N-HA-TGFBI转染组、空质粒pCMV—N—HA转染组和阴性对照组间角膜上皮的吸光度（A450）值的差异无统计学意义（F=3．34，P〉0Background The human transforming growth factor beta-induced gene （TGFBI） is the first determined pathogenic gene to corneal dystrophy. But the molecular genetic mechanism is completely unknown. The study of concerning role of TGFBI is very important for us understand the physiological function of cornea, and the pathogenesis of corneal dystrophy. Objective The vector of human transforming growth factor beta-induced gene （TGFBI） in eukaryotic expression was constructed and transfected into the human corneal epithelial ceils in order to explore its influence on the growth of human corneal epithelial cells. Methods Total RNA was extracted from normal donor cornea tissue and cDNA was obtained by reverse transcription. TGFBI eDNA was synthesized by reverse transcription-PCR and cloned into pCMV-N-HA vector and identified by sequencing with PCR and EcoRV, XhoI double restriction endonuclease. The cells were grouped into recombinant pCMV-N-HA-TGFBI plasmid group,pCMV- N-HA plasmid group, non-transfected group and pGFP-C2 transfected group. The recombinant pCMV-N-HA-TGFBI plasmid was transfected to human corneal epithelial cells and identified by observing the expression of enhanced green fluorescence protein（ EGFP） in the cells. The TGFBI mRNA and proteins were harvested from the cells for real-time PCR analysis and Western blot assay respectively in 58 hours after transfection. The growth of the transfected cells was assessed by Cell Counting Kit-8. The expressions of matrix metalloproteinase （MMP）and tissue inhibitors of matrix metalloproteinase （TIMP） proteins and their mRNA in transfected ceils were detected using SYBR fluorescence realtime PCR analysis and Western blot assay. Results The sequencing result of pCMV-N-HA-TGFBI positive clone plasmid showed that amplified TGFBI eDNA inserted into the vector at the correct sequence. EGFP was expressed in transfected cells in 48 hours after transfer of pGFP-C2 with the transfer efficacy 70%. The expression intensity of TGFBI mRNA was significantly hi

关 键 词：转化生长因子β诱导基因 角膜上皮细胞 基质金属蛋白酶 真核表达载体 

分 类 号：R772.2[医药卫生—眼科] Q812[医药卫生—临床医学]