摘要
构建人cyclinG2基因真核表达载体,进一步研究cyclinG2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cyclinG2基因cDNA,成功构建了真核表达载体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cyclinG2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,p21WAF1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cyclinG2抑制细胞增殖作用可能是通过诱导p21WAF1的表达而实现。
In order to make sure whether expression of eyclin G2 gene promotes or inhibits cell proliferation in vitro, and to investigate its mechanism. The whole length of eyclin G2 cDNA was cloned by reverse transcription- polymerase chain reaction （RT-PCR）, and was inserted into plRESneo eukaryotie expression vector, testified pIRES-G2 recombinant plasmid was constructed successfully. The construct was then introduced into HeLa and CV-1 cells in order to observe the effect of cyelin G2 transgene expression on the colony forming capacity, and protein expression of p21^WAF1 in transfeetant cells was examined by cellular immunochemieal staining. The results showed that colony-forming efficiency of the cells transfected with pIRES-G2 construct was much lower compared with the control parental vector plRESneo. Furthermore, pIRES-G2 transfeeted 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 transfeeted with pIRES-G2 plasmid DNA showed positive p21^WAF1 staining and labeling intensity increased significantly compared with its corresponding transfeetant cells with the empty control vector （p 〈 0.01 ）. So it was concluded that eetopie over expression of eyclin G2 inhibit in vitro cell proliferation of cancer and normal cells and it may negatively regulate the cell cycle by upregulating p21^WAF1 expression .
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2006年第6期30-35,共6页
China Biotechnology
基金
教育部高等学校优秀青年教师科研奖励计划资助项目(2000-26)
