人细胞周期蛋白G2基因真核表达载体构建及其功能研究

中国生物工程杂志

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人细胞周期蛋白G2基因真核表达载体构建及其功能研究

田玉楼刘洁张学

中国医科大学口腔医学院正畸科中国医科大学实验技术中心中国医科大学卫生部细胞生物学重点实验室医学基因组学研究室

Construction of Human Cyclin G2 Gene Eukaryotic Expression Vector and Research of its Function

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摘要： 构建人cyclin G2基因真核表达载体，进一步研究cyclin G2对体外培养细胞增殖的调节作用及可能的调节机制。以人口腔癌前上皮细胞系POE4总RNA的反转录产物为模板，应用RT-PCR方法克隆cyclin G2基因cDNA，成功构建真核表达载体pIRES -G2；应用脂质体介导的基因转染技术，以体外培养的肿瘤细胞系HeLa细胞和正常细胞系CV-1细胞作为受体细胞，进行转基因表达研究，发现cyclin G2高表达对体外培养细胞的增殖起明显抑制作用；应用p16INK4a、p21WAF1、p27KIP1三种周期蛋白依赖性激酶抑制因子的单克隆抗体对转基因的HeLa细胞进行免疫细胞化学研究，发现转染pIRES-G2的实验组细胞中，p21 WAF1蛋白染色阳性细胞数明显多于转染空载体的对照组，平均光密度值高于对照组，两组间均有显著性差异（p<0.01），提示cyclin G2抑制细胞增殖作用可能是通过诱导p21WAF1的表达而实现。

Abstract: In order to make sure whether expression of cyclin G2 gene promotes or inhibits cell proliferation in vitro, and to investigate its mechanism. we have cloned the whole length of cyclin G2 cDNA by reverse transcription - polymerase chain reaction (RT-PCR ) , and inserted it into pIRESneo eukaryotic expression vector, testified pIRES-G2 recombinant plasmid constructed successfully. The construct was then introduced into HeLa and CV-1 cells in order to observe the effect of cyclin G2 transgene expression on the colony forming capacity, and protein expression of p21WAF1 in transfectant cells was examined by cellular immunochemical staining. The results showed that colony-forming efficiency of the cells transfected with pIRES-G2 construct was much lower compared with that with the control parental vector pIRESneo. Furthermore, pIRES-G2 transfected HeLa cells showed a senescent morphology. The experimental group of CV-1 cells could hardly form any detectable colony, while the corresponding empty vector group showed large patches of colony. More HeLa cells transfected with pIRES-G2 plasmid DNA showed positive p21WAF1 staining and labeling intensity increased significantly compared with its corresponding transfectant cells with the empty control vector (p<0.01). So we conclude that ectopic over expression of cyclin G2 inhibit in vitro cell proliferation of cancer and normal cells and it may negatively regulate the cell cycle by upregulating p21WAF1 expression .

收稿日期: 2006-02-23 出版日期: 2006-06-25

通讯作者: 田玉楼

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引用本文:

田玉楼,刘洁,张学. 人细胞周期蛋白G2基因真核表达载体构建及其功能研究[J]. 中国生物工程杂志, .

. Construction of Human Cyclin G2 Gene Eukaryotic Expression Vector and Research of its Function. China Biotechnology, .

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https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2006/V26/I06/30

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