靶向钠钾ATP酶α1亚单位shRNA质粒表达载体的构建与筛选  

Construction and Screening of Plasmid Expression Vectors Encoding the Short Hairpin RNA Targeting Na^+/K^+-ATPase α_1 Subunit

作　　者：周蔚[1,2] 徐忠伟[1,2] 王凤梅[3] 陈小义[1,2] 呼文亮[2] 徐瑞成[2] 

机构地区：[1]中国人民武装警察部队医学院细胞生物学与遗传学教研室,天津300162 [2]中国人民武装警察部队医学院天津市职业与环境危害生物标志物重点实验室,天津300162 [3]天津市第三中心医院肝内科,天津300171

出　　处：《生物技术通报》2010年第11期212-218,共7页Biotechnology Bulletin

基　　金：国家自然科学基金(0840010)

摘　　要：构建编码人钠钾ATP酶(Na+/K+-ATPase)α1mRNA的短发夹RNA(shRNA)质粒表达载体shRNA-ATP1A1(ATP1A11、ATP1A12和ATP1A13),并筛选出基因沉默效果最明显的shRNA质粒表达载体。设计、合成靶向ATP1A1的3对DNA序列,分别插入Pgenesil-3中构建3个shRNA表达载体,经限制性内切酶酶切和DNA测序鉴定确认。筛选并确定最佳细胞接种量及重组质粒转染量,半定量逆转录聚合酶链反应(RT-PCR)和免疫细胞荧光检测Na+/K+-ATPaseα1表达变化;MTT法和流式细胞术检测沉默效果最明显的ATP1A13对HepG2增殖活性和细胞周期的影响。构建的质粒表达载体酶切鉴定均可扩增出预期条带,测序符合设计要求,构建成功。ATP1A12和ATP1A13对所转染的HepG2细胞中Na+/K+-ATPaseα1mR-NA和蛋白质表达均有抑制作用,其中ATP1A13最为明显(P<0.05)。ATP1A13可抑制HepG2细胞的增殖;转染48和60h,HepG2细胞细胞周期呈现S期阻滞;实时定量PCR检测ATP1A13敲低HepG2Na+/K+-ATPaseα1mRNA呈时间依赖性,72h后表达降低约90%。试验成功构建靶向钠钾ATP酶α1亚单位的shRNA质粒表达载体,其中shRNA-ATP1A13可显著抑制HepG2细胞增殖引起细胞周期S期阻滞。It was to construct the short hairpin RNA（shRNA）expressing vector shRNA-ATP1A1（ATP1A11,ATP1A12,ATP1A13）targeting human Na^＋/K^＋-ATPase α1 mRNA.The hepatocellular carcinoma HepG2 cell line were trasfected with vector shRNA-ATP1A1 for 24 h.The Na^＋/K^＋-ATPase α1 subunit expression level of HepG2 were detected by RT-PCR and immunocytochemistry.The shRNA-ATP1A13 could significantly knock down the Na^＋/K^＋-ATPase α1 subunit expression of HepG2 cells compared to the ATP1A11 and ATP1A12 group（P〈0.05）.The proliferation activity of HepG2 treated with shRNA-ATP1A13 was determined by MTT assay,cell cycle was measured by flow cytometry and Na^＋/K^＋-ATPase α1 mRNA level were determined by Real-time PCR.We found that shRNA-ATP1A13 could inhibit HepG2 cell proliferation in time dependent manner.Cell cycle showed S phase arresting after transfecting for 48 and 60 h.Real-time PCR result showed that the knockdown effect of Na^＋/K^＋-ATPase α1 mRNA expression level were in time dependent manners,and it decreased about 90 % after 72 hours.The plasmid expression vectors coding for shRNA targeting Na^＋/K^＋-ATPase α1 mRNA have been constructed successfully,and shRNA-ATP1A13 could inhibit HepG2 cell proliferation and induce cell cycle S phase arresting.

关 键 词：钠钾ATP酶 RNA干扰 HEPG2细胞 细胞周期 

分 类 号：Q78[生物学—分子生物学]