胶质细胞生长因子真核表达载体的构建及其在脊髓内的表达  

Construction of glial growth factor eukaryotic expression vector and its expression in spinal cord

作　　者：魏梅洋[1] 董艳[2] 李家顺[1] 贾连顺[1] 薛亚军[2] 韩晞[2] 

机构地区：[1]解放军第二军医大学长征医院骨科,上海市200003 [2]解放军第二军医大学长征医院神经外科,上海市200003

出　　处：《中国临床康复》2005年第41期54-56,i0002,共4页Chinese Journal of Clinical Rehabilitation

摘　　要：目的:构建胶质细胞生长因子2真核表达载体pEGFP-N1-GGF2,观察其在大鼠脊髓内的表达。方法:实验于2004-01／10在解放军第二军医大学长征医院神经外科实验室完成。出生1周SD大鼠2只和体质量250-300 g雄性成年SD大鼠12只。将成年大鼠分为对照组和实验组,每组6只。①采用反转录聚合酶链反应方法从大鼠胎脑组织总RNA中扩增出胶质细胞生长因子2 的全序列cDNA。②以融合蛋白方式克隆到增强型绿色荧光蛋白报告基因的真核表达载体pEGFP-N1中,构建重组质粒pEGFP-N1-GGF2。③采用基因注射法将阳离子脂质体Transfectam和pEGFP-M1-GGF2混合后转染至实验组大鼠胸段脊髓组织中;对照组大鼠只注射脂质体和空白质粒pEGFP-N1的混合物。基因注射后1周,两组各取3只大鼠麻醉后取材行反转录聚合酶链反应,另取3只大鼠麻醉后取出T8,T9脊髓,冰冻切片后行荧光照相,观察绿色荧光蛋白表达情况。结果:12只大鼠均进入结果分析。①大鼠胶质细胞生长因子2 cDNA的克隆结果:成功克隆胶质细胞生长因子2 cDNA。②重组质粒pEGFP- N1-GGF2的构建结果:酶切鉴定及测序分析证实成功构建胶质细胞生长因子真核表达载体pEGFP-N1-GGF2。③反转录聚合酶链反应证明胶质细胞生长因子mRNA表达:实验组基因转染1周后局部脊髓内胶质细胞生长因子2 mRNA的表达显著增加。④pECFP-N1-GGF2基因体内转染结果:实验组注射局部脊髓内灰质和白质神经细胞内均有较多绿色荧光蛋白表达;而对照组未见荧光蛋白表达。结论:阳离子脂质体介导胶质细胞生长因子2基因体内转染的方法是可行的,增强型绿色荧光蛋白可作为报告基因观察胶质细胞生长因子2 基因在体内的表达。AIM： To construct glial growth factor eukaryotic expression vector of pEGFP-N1-GGF2, and observe its expression in the spinal cord. METHODS： The experiment was finished in the laboratory of Department of Neurosurgery, Changzheng Hospital of the Second Military Medical University of Chinese PLA between January and October 2004, Two 1-week-old SD rats and 12 adult male SD rats of 205-300 g were used. The adult rats were divided into control group （n=6） and experimental group （n=6）. ①The eDNA encoding the glial growth factor 2 （GGF2） was isolated by using reverse transcription-polymerase chain reaction （RT-PCR） with total RNA extracted from fetal rat brain. ② GGF2 eDNA was cloned into eukaryotic expression vector pEGFP N1 of enhanced green fluorescent protein （EGFP） reported gene encoding green fluorescence protein in the form of fused protein, The expression vector of recombinant plasmid pEGFP-N1- GGF2 was successfully constructed. ③ The recombinant plasmid pEGFP-N 1-GGF2 and transfectam liposome complexes were injected into adult rats thoracic spinal cord, and the rats in the control group were only injected with the mixture ofliposomes and blank plasmid pEGFP-N1. One week after injection, 3 rats in each group were anesthetized to remove materials for RT-PCR, and the other 3 were anesthetized to remove T8 and T9 spinal cords, and then fluorescent photography was conducted, and the expression of green fluorescent protein was observed. RESULTS： All the 12 rats were involved in the analysis of results.① Results of GGF2 eDNA clone： GGF2 cDNA were successfully cloned, ② Results of recombinant plasmid pEGFP-N1-GGF2： The analysis by restricting enzyme digestion and DNA sequencing showed that GGF2 cDNA fragment was cloned into pEGFP-N1. ③ The expression of GGF2 mRNA confirmed by RT-PCR： The expression of GGF2 mRNA in local spinal cord at 1 week ＇after gene transfection was significantly increased in the experimental group. ④Results of pEGFP-N 1-GGF2 gene transfeetion in vivo

关 键 词：受体 生长因子 逆转录聚合酶链反应 重组融合蛋白质类 转染 

分 类 号：R651.21[医药卫生—外科学]