p38丝裂原激活蛋白激酶基因重组慢病毒载体的构建及其在建立人前列腺癌稳定细胞株中的应用  

Construction of a recombinant lentiviral vector of p38 MAPK and establishment of a human prostatic carcinoma cell line stably expressing p38 MAPK

作　　者：荆玉明[1] 罗杰[2] 张艳玲[2] 陈三三[1] 万沛[1] 颜仁和[2] 王宏昌[2] 陈白虹[2] 谭万龙[1] 李红卫[2] 

机构地区：[1]南方医科大学南方医院泌尿外科,广东广州510515 [2]南方医科大学生物技术学院,广东广州510515

出　　处：《南方医科大学学报》2012年第3期317-321,共5页Journal of Southern Medical University

基　　金：国家自然科学基金(81072113)~~

摘　　要：目的构建p38丝裂原激活蛋白激酶基因(MAPK)重组慢病毒载体并建立表达外源性p38基因的人前列腺癌稳定细胞株。方法采用限制性内切酶酶切、T4 DNA连接酶连接等方法,将EGFP/p38融合基因插入慢病毒载体pTYF-EF1α-IRES-EGFP中,构建启动子EF1α调控的EGFP/p38共表达慢病毒载体pTYF-EF1α-EGFP/p38,经酶切鉴定后,利用慢病毒三质粒系统,通过脂质体法转染包装细胞HEK 293T细胞,收集病毒上清后转导人前列腺癌DU145细胞株,经有限稀释法筛选重组EGFP/p38稳定细胞株,用Western blotting检测细胞总p38的表达,用计数法绘制细胞的生长曲线。结果经酶切鉴定,成功构建EGFP/p38重组慢病毒载体,并包装慢病毒,检测病毒悬液的滴度为4.7×106 TU/ml,利用此病毒悬液感染DU145细胞,成功筛选到EGFP/p38稳定表达细胞株,Western blotting显示该细胞株能稳定表达外源性EGFP/p38融合蛋白,这种过表达p38的细胞株的生长速度明显低于对照组。结论成功构建p38 MAPK重组慢病毒载体并建立了表达外源性p38 MAPK基因的稳定细胞株EGFP/p38-DU145,细胞内p38过表达能够抑制DU145细胞的增殖。Objective To construct a recombinant lentiviral vector for p38 MAPK and establish a human prostatic carcinoma cell line that stably expresses p38 MAPK.Methods EGFP/p38 fusion gene was subcloned into the lentiviral vector pTYF-EF1α-IRES-EGFP.The recombinant lentiviral vector pTYF-EF1α-EGFP/p38 was indentified by restriction enzyme digestion,and packaged in HEK 293T cells using lipofectamintm2000 with the packaging plasmid psPAX2 and envelope plasmid pMD2.G.The viral titer was tested according to the expression level of GFP.The resulting recombinant lentiviral vector was transduced into human prostatic carcinoma DU145 cells,and stably transduced cells were selected by limiting dilution analysis.The intracellular expression level of total p38 was detected by Western blotting and the cell growth curve was drawn.Results DNA restriction enzyme digestion demonstrated that the recombinant lentiviral vector of the fusion gene EGFP/p38（pTYF-EF1α-EGFP/p38） was constructed successfully.The recombinant lentiviral vector was packaged in 293T with a viral titer of 4.7×106 TU/ml.A stable cell line,EGFP/p38-DU145,was established,which stably expressed exogenous EGFP/p38 MAPK fusion protein as detected by Western blotting and showed a lowered growth rate compared to the control cells.Conclusion We have successfully constructed a recombinant lentiviral vector of the fusion gene EGFP/p38 and established a stable cell line EGFP/p38-DU145.Overexpression of p38 has a significant inhibitory effect on the proliferation of DU145 cells in vitro.

关 键 词：P38丝裂原激活蛋白激酶 稳定细胞株 前列腺癌 慢病毒 

分 类 号：R737.25[医药卫生—肿瘤] R373[医药卫生—临床医学]