ATP1B1真核表达质粒的构建及其对胃腺癌SGC-7901细胞的作用  

Construction of Eukaryotic Expression Plasmid pEGFP-ATP1B1 and Its Effect on Gastric Adenocarcinoma Cell SGC-7901

作　　者：熊竹娟[1] 林苹[1] 张洁[1] 王修杰[1] 王琪[1] 任婧婧[1] 杨洪亮[1] 王静[1] 吴亚英[1] 

机构地区：[1]四川大学华西医院生物治疗国家重点实验室实验肿瘤研究室,成都610041

出　　处：《四川大学学报（医学版）》2008年第2期169-172,176,共5页Journal of Sichuan University(Medical Sciences)

基　　金：华西医院回国人员启动基金资助

摘　　要：目的构建Na+,K+-ATP酶β1亚基(ATP1B1)真核表达质粒,为利用ATP1B1进行肿瘤基因治疗奠定基础。方法以白细胞文库为模板扩增ATP1B1 cDNA,定向插入到pEGFP-C3真核表达质粒中,构建pEGFP-ATP1B1重组质粒;经酶切分析和测序鉴定后,通过脂质体介导转染胃腺癌SGC-7901细胞,利用real-time PCR检测转染后SGC-7901细胞的ATP1B1基因表达,并进行其ATP酶活性检测;MTT法测定转染后SGC-7901细胞的增殖活性。结果重组质粒经鉴定证实含有ATP1B1 cDNA序列,转染pEGFP-ATP1B1的SGC-7901细胞ATP1B1基因表达增高达129.2%,ATP酶活性增强为(2.95±0.210)%,细胞生长增殖显著受抑。结论成功构建了ATP1B1真核表达质粒,为进一步利用ATP1B1研究恶性肿瘤基因治疗奠定基础。Objective To establish the eukaryotic expression plasmid containing the code gene of Na^＋,K^＋- ATPase β1-subunit （ATP1B1） and the basis of ATP1B1 applied to antitumor gene therapy. Methods The ATP1B1 cDNA was amplified from leukocyte gene library and then cloned into the eukaryotic expression vector pEGFP-C3. The recombinant plasmid, named as pEGFP-ATP1B1, was determined with restriction enzyme and sequencing analyses. Next pEGFP-ATP1B1 was transferred into gastric adenocarcinoma SGC-7901 cells by lipofectamine, then ATP1B1 mRNA expression in transfected cells was detected by real-time PCR, and also ATPase was detected after cell transfection,as well as the proliferation of such cells was measured by MTT. Results The analysis confirmed that the recombinant pEGFP-ATP1B1 contained the ATP1B1 cDNA. After cell transfection, the expression of ATP1B1 mRNA（129.2%） and the activity of ATPase [（2.95±0. 210）%] were higher, and the growth of the SGC-7901 cells transfected with ATP1B1 was inhibited obviously when compared with the control group. Conclusion The recombinant pEGFP-ATP1B1 is constructed successfully, and this recombinant eukaryotic expression vector could be used in additional studies on the biological effect of ATP1B1 and its use in anti-tumor gene therapy.

关 键 词：Na^+ K^+-ATP酶β1亚基 胃腺癌SGC-7901细胞 转染 肿瘤基因治疗 

分 类 号：R735.2[医药卫生—肿瘤]