小鼠Ghrelin基因真核表达质粒的构建及对3T3-L1前脂肪细胞增殖的影响  

Construction of a eukaryotic expression plasmid with mouse Ghrelin protein gene and its effect on the proliferation of 3T3-L1

作　　者：陈健[1] 叶亚琼[1] 欧阳丹[1] 马浩然[1] 马勇江[1] 张媛[1] 李玉谷[1] 

机构地区：[1]华南农业大学兽医学院,广东广州510642

出　　处：《中国兽医科学》2015年第12期1300-1306,共7页Chinese Veterinary Science

基　　金：国家自然科学基金资助项目(31272519)

摘　　要：为构建小鼠Ghrelin基因的真核表达质粒,并研究Ghrelin对3T3-L1细胞增殖的影响,以小鼠肠cDNA为模板,通过PCR扩增Ghrelin基因,插入到pMD18-T克隆载体中。对经鉴定的重组质粒pMD-Ghrelin进行双酶切,将目的基因连接到经同样双酶切的真核表达载体pcDNA3.1(+)上。重组质粒经双酶切分析和测序鉴定后,用Lipofectamine 3000转染试剂将重组表达质粒转染到3T3-L1细胞中,采用qRT-PCR和Real-time PCR检测Ghrelin基因在3T3-L1细胞中的表达。采用CCK-8法检测细胞的生长活力;流式细胞术检测Ghrelin基因对细胞周期和凋亡的影响;采用Real-time PCR检测部分周期相关基因的mRNA表达水平。结果显示,成功构建了小鼠Ghrelin基因的真核表达质粒pcDNA3.1(+)-Ghrelin,并能在3T3-L1细胞中得到很好地表达,其表达倍数是空质粒组的5倍(P<0.01);转染后第48、72和96小时,Ghrelin转染组3T3-L1细胞的生长活力明显高于空载体组(P<0.05或<0.01);转染后第24小时,Ghrelin转染组S期细胞数量明显高于空载体组(P<0.05);Ghrelin过表达对3T3-L1细胞的凋亡无明显影响(P>0.05)。qRT-PCR结果显示,Ghrelin转染组细胞的c-myc(P<0.05)、E2F1、CDK2(P<0.05)等mRNA的表达量也明显高于空载体组,P53表达低于空载体组,差异显著(P<0.05)。以上结果表明,Ghrelin过表达能显著促进3T3-L1细胞的增殖,但不影响凋亡。To construct a recombinant eukaryotic plasmid with mouse Ghrelin protein gene and to study the effect on proliferation of 3T3-L1 cells,Ghrelin gene was amplified by PCR from cDNA of mouse small intestine.The amplified Ghrelin gene was cloned into pMD18-T and the recombinant pMD-Ghrelin plasmid was extracted.The plasmid was digested with the double enzymes and the objective genes were connected with pcDNA3.1（＋）which had been digested with the same enzymes.After the recombinant plasmid was identified by double restriction enzyme digestion and gene sequence analysis,3T3-L1 cells were transfected with the recombinant plasmid by Lipofectamine 3000.The expression of Ghrelin gene in 3T3-L1 cells was analyzed by qRT-PCR and real-time PCR.In result,the eukaryotic expression plasmid pcDNA3.1（＋）-Ghrelin was constructed successfully in 3T3-L1 cells.It is five times higher than control.At hour 48,72 and 96post-transfection,Ghrelin can promote 3T3-L1proliferation（P0.05or0.01）.At hour 24,the cell content of pcDNA3.1（＋）-Ghrelin is higher than pcDNA3.1（＋）in S phase（P0.05）.It is no obvious difference in ghrelin overexpression in 3T3-L1cells（P0.05）.qRT-PCR demonstrated that the expression quantity of pcDNA3.1（＋）-Ghrelin,c-myc（P0.05）,E2F1,CDK2（P0.05）mRNA is higher than pcDNA3.1（＋）.However,P53（P0.05）mRNA is lower than control.Results showed that the overexpression of Ghrelin could significantly promote the proliferation of 3T3-L1 cells,but no apoptosis.

关 键 词：GHRELIN基因 真核表达质粒 3T3-L1细胞 细胞增殖 小鼠 

分 类 号：Q786[生物学—分子生物学]