PRL-2基因转染对肝细胞增殖及细胞周期的影响  

Effect of PRL-2 gene transfection on proliferation and cell cycle in hepatocellular cell line

作　　者：程超[1] 郭爱林[2] 巫国勇[1] 吴伟康[3] 罗红鹤[1] 钟佛添[1] 马俊[1] 何伟玲[1] 

机构地区：[1]中山大学附属第一医院 [2]广东省人民医院医学研究中心 [3]中山大学基础医学院病理生理教研室,广东广州510080

出　　处：《中国病理生理杂志》2007年第11期2229-2233,共5页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No30100218);广东省科技厅资助项目(No2004B30301010;No2005B30301021)

摘　　要：目的:研究PRL-2基因对肝细胞增殖和细胞周期的影响。方法:采用脂质体转染的方法将重组质粒稳定转染至正常永生化肝细胞系CL1中,G418筛选阳性克隆。应用实时荧光定量聚合酶链反应、Western印迹和免疫组化分析PRL-2在阳性细胞的表达及蛋白定位,MTT法检测细胞的群体倍增时间,流式细胞仪检测细胞周期变化,Western印迹分析细胞周期素A、D1、E以及周期蛋白依赖激酶抑制因子p16、p21WAF1及p27Kip1的变化,实时荧光定量聚合酶链反应检测p21WAF1 mRNA的变化。结果:成功构建PRL-2真核表达载体pcDNA3-PRL-2。脂质体转染细胞经过G418筛选后,获得稳定表达PRL-2的细胞亚系PRL-2-CL1。经实时荧光定量PCR、Western印迹和免疫组化证实,PRL-2-CL1细胞系PRL-2基因及蛋白表达水平高于对照组,流式细胞仪检测细胞周期S期细胞比例明显增高,MTT法检测细胞群体倍增时间缩短。Western印迹及实时荧光定量聚合酶链反应显示p21WAF1在转染后较对照组明显降低,细胞周期素A、D1、E及p16、p27Kip1无明显变化。结论:重组PRL-2真核表达载体构建正确,并在永生化肝细胞中获得稳定、高效表达。PRL-2基因具有促进细胞增殖的作用,这种作用与其降低p21WAF1蛋白含量相关。AIM ： To observe the changes of proliferation and cell cycle after PRL - 2 gene effectively expressed in human hepatocellular cell line. METHODS： The PRL - 2 vector was transfected into CL1 cell with lipo-fectamine reagent, the stable expression clones were screened by G418. The expression of PRL - 2 mRNA was detected by real -time PCR. The expressive protein was identified by Western blotting. The subcellular localization was demonstrated by immunochemistry. The cell cycle was measured by flow cytometry. The population doubling time （TD） was analyzed by MTT assay. The expressions of cyclin A, cyclin D1, cyclin E, pl6, p21^WAF1 and p27^Kip1 were detected by Western blotting. The p21^WAF1 mRNA was determined by real - time PCR. RESULTS： The full length ORF of PRL -2 gene was inserted in-to the vector pcDNA3. 1 （ ＋ ）, transfected into CL1 cells, and expressed successfully. Real - time PCR showed stable expression of PRL -2 mRNA. Western blotting confirmed the overexpression of PRL -2 protein. The subcellular localization of PRL - 2 was in the plasmid. The proportion of cells in S - phase was increased. The population doubling time was reduced （ P 〈 0. 01 ）, a significant decrease was observed both in the mRNA and the protein expression of the p21^WAF1 in com- parison with untransfected or vector - transfected control cells （ P 〈 0. 05）. The expressions of cyclin D1, cyclin E, cycli- nA, pl6 and p27^Kip1 were not appreciably different between the control and PRL - transfected cell lines. CONCLUSION： Eukaryocytic expression vector of PRL - 2 has been successfully constructed, which shows stable and effective expression in CL1 cell line. PRL -2 increases cell proliferation by stimulating progression from G1 into S phase, which is primarily associated with decreased p21^WAF1.

关 键 词：肝细胞 蛋白质酪氨酸磷酸酶 真核表达载体 细胞周期 

分 类 号：R392.11[医药卫生—免疫学]