小鼠Nanog基因的克隆及对人宫颈癌上皮细胞的作用  

Cloning of mouse Nanog gene and its effect on human Hela Cells

作　　者：窦琳[1] 吕长荣[1] 李军[1] 贾文文[1] 赵婷[1] 窦忠英[1] 

机构地区：[1]西北农林科技大学陕西省干细胞研究中心,陕西省杨凌市712100

出　　处：《中国组织工程研究与临床康复》2007年第7期1239-1242,I0003,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家"八六三"计划资助项目(2005AA21905);教育部重大项目资助(0316)~~

摘　　要：目的:克隆小鼠Nanog基因并构建带绿色荧光蛋白的真核表达载体pG-Nanog,观察其对人宫颈癌上皮细胞(Hela细胞)中的表达,旨在为进一步观察其对成体细胞的表型变化及细胞增殖奠定前期实验学基础。方法:实验于2006-03/09在西北农林科技大学陕西省干细胞研究中心完成。Nanog基因的克隆参照庄淑珍的方法。Nanog基因真核表达载体的构建参照GeneBank中的小鼠Nanog基因序列,以pNA992为模板扩增Nanog基因,PCR产物以BglⅡ和SacⅡ双酶切,同时将pEGFP-C1用BglⅡ和SacⅡ鉴定,将该质粒命名为pG-Nanog。Hela细胞用含10%新生牛血清的Dulbecco’s改良培养基(Dulbecco’sModifiedEagleMedium,DMEM)培养,转染Hela细胞。转染前1d,在6孔板的每个孔中接种1.2×105个细胞,待细胞生长至60%～70%汇合时,取pG-Nanog与空载体各4μg分别加入500μL无血清无抗生素的DMEM培养液中,同时将6μL稀释于500μL无血清无抗生素的DMEM(干粉)培养液中,将两者混合,室温静置20min,将复合物加入到细胞中,置37℃,体积分数0.05的CO2培养箱中转染24h后吸出复合物加入完全培养基,48h后观察荧光。合成内源对照β-actin,收集转染4d后的细胞提取RNA,反转录为cDNA,然后分别扩增Nanog基因和β-actin基因。采用RT-PCR的方法检测Hela细胞中Nanog基因的表达。结果:①pEGFP-C1载体经双酶切后获得约1kbp的Nanog基因真核表达载体片段,同预期结果相一致,测序结果同GeneBank中的序列同源性达到99.7%。②将转染48h后的Hela细胞置于荧光显微镜下观察,可见明显的荧光,转染pG-Nanog的细胞绿色荧光蛋白集中于细胞核,将转染4d后Hela细胞的总RNA进行RT-PCR检测,产物经琼脂糖电泳分析,只有转染pG-Nanog的细胞中才能够检测到Nanog基因的相应条带。③转染48h后,对细胞进行抗增殖细胞核抗原免疫组化染色,未转染细胞和转染空质粒细胞及转染pG-Nanog细胞染色结果均呈阳性,转染了pG-Nanog的HelaAIM： To clone Nanog gene in mouse, construct Nanog expression vector pG-Nanog with green fluorescent protein （GFP） and express it in Hela cells. This will lay a foundation for further study of phenotype of adult cells and cell proliferation. METHODS： The experiment was carried out in the Shaanxi Branch of National Stem Cell Engineering and Technology Center from March to September 2006. RT-PCR was used to clone Nanog gene from mRNA of mouse ESCs. A pair of primer was designed according to the sequence of Nanog gene published in GeneBank. The DNA fragment of Nanog gene was amplified by PCR from the pNA992 recombinant plasmid. After being confirmed by sequencing, the Nanog gene was inserted into the pEGFP-C1 vector to construct expression vector pG-Nanog, which was evaluated by Bgl Ⅱ and Sac Ⅱ double enzyme restriction. Hela cells was cultured in Dulbecco＇s Modified Eagle Medium （DMEM） containing 10% new bovine serum, and were seeded at 1.2×10^5 per well in six-well plates. When the cultures were 60%-70% confluent, the plasmid pG-Nanog was transfected into Hela cells. In brief, 4 μg DNA was incubated with 500 μL DMEM without serum, and 6 μL Lipofectamine 2000 was incubated with 500 μL DMEM without serum. Afterwards both of them were mixed together and placed at room temperature for 20 minutes. The mixture was added to the transfected cells that were cultured in 5% CO2 incubator at 37 ℃ for 24 hours, then fed with fresh medium. After 48 hours, the green fluorescence of cells was visualized under a UV microscope. Endogenous control β-actin was synthesized, cells were harvested Io extract mRNA and then transfer into cDNA. RT-PCR was used to detect the expression of Nanog gene in Hela cells. RESULTS： （1）A 1 kbp fragment of Nanog gene expression vector was obtained by digesting pEGFP-C1 with Bgl Ⅱ and Sac Ⅱ .The sequence of the cloned Nanog was confirmed to be correct by sequence homology analysis, accounting for 99.7%.（2）The Hela .cells transfected with pG-Nanog were cultured for

关 键 词：基因 真核细胞 基因表达 HELA细胞 

分 类 号：R737.33[医药卫生—肿瘤]