过表达DNMT3B7质粒载体的构建及对293A细胞增殖的影响  

Construction of DNMT3B7 Eukaryotic Expression Vector and Its Effect on Proliferation of 293A Cells

作　　者：张姝[1,2] 张伟[3] 姜树原[1,2] 张颜波[4] 吴松泉[5] 黄丽华[1,2] 孟宪梅[3] 吉训明[6,7] 邵国[1,2,6,7] 

机构地区：[1]包头医学院生物医学研究中心,内蒙古包头014010 [2]包头医学院基础医学院 [3]包头医学院第二附属医院消化科 [4]泰山医学院附属医院神经内科 [5]丽水学院医学院 [6]首都医科大学宣武医院神经内科 [7]首都医科大学宣武医院 神经再生教育部重点实验室

出　　处：《肿瘤防治研究》2013年第12期1128-1133,共6页Cancer Research on Prevention and Treatment

基　　金：国家自然科学基金资助项目(81060212;81160244;81360316);中国博士后基金资助项目(20080430851);内蒙古自然科学基金资助项目(2010BS1104);内蒙古自治区高等学校科研项目资助课题(NJZY12221;NJZY12225;NJYT-13-A10)

摘　　要：目的构建DNA甲基转移酶3B(DNMT3B)的一种异构体3B7的真核质粒表达载体,在体外评价其对人胚肾细胞株293A增殖影响。方法据GenBank中人DNMT3B7 cDNA序列设计并合成特异性引物,以DNMT3B1为模板,利用高保真Taq酶,进行PCR技术扩增DNMT3B7,并将扩增产物克隆到真核表达载体pCMV-2B中。将携带DNMT3B7基因的质粒pCMV-DNMT3B7及空质粒pCMV-2B转染293A细胞,通过G418筛选出稳定表达的细胞系,MTT法检测细胞的增殖;流式细胞仪检测细胞的细胞周期分布。同时检测p21蛋白表达情况。结果成功构建DNMT3B7的真核表达载体并筛选出稳定表达株,与转染空质粒组相比,稳定过表达DNMT3B7组293A细胞株增殖减慢(P<0.01);过表达DNMT3B7的293A细胞S期细胞比例显著增多(P<0.05)。p21 mRNA和蛋白表达增加(P<0.05)。结论 DNMT3B7基因过表达可抑制293A细胞增殖,p21可能参与了这一过程的调节。Objective The aim of this study was to construct eukaryotic expression plasmid for human pCMV-DNA methyltransferase 3B7 （DNMT3B7） and investigate the effects of over-expression of DNMT 3B7 gene on proliferation of human embryo kidney 293A cells. Methods DNMT3B7 was amplified by PCR using highfidelity Taq and cloned into pCMV-2B. 293A cells were transfected with plasmid pCMV-DNMT 3B7 or pCMV-2B） and then treated with G418 to get the stable cell lines. The proliferation level of stable cell lines was determined by MTT, and cell cycle distribution by flow cytometry. Results DNMT3B7 eukaryotic expression plasmid was successfully constructed. Compared with those 293A cells transfected with pCMV- 2B, the number of those cells with over-expression DNMT 3B7 was decreased （P〈0.01）. The percentage of 293A cells with overexpression DNMT 3B7 at S stage was decreased （P〈0.05）. The mRNA level and pro- tein level of p21 were increased （P〈0.05）. Conclusion The over-expression of DNMT 3B7 gene inhibited the proliferation of 293A, maybe through p21.

关 键 词：DNA甲基转移酶3B 293A细胞 细胞增殖 P21 

分 类 号：Q291[生物学—细胞生物学]