靶向IRE1a干扰质粒构建及对ERS时细胞增殖及凋亡的影响  

IRE1a-shRNA Plasmid Construction and Its Effect on Cell Proliferation and Apoptosis

作　　者：姜蓉[1] 刘艳娜[2,3] 周菁华[2,3] 刘平[2,3] 郭风劲[2,3] 

机构地区：[1]重庆医科大学干细胞与组织工程研究室,重庆市400016 [2]重庆医科大学细胞生物学及遗传学教研室,重庆市400016 [3]重庆医科大学发育生物学与模式动物平台,重庆市400016

出　　处：《医学分子生物学杂志》2012年第2期103-109,共7页Journal of Medical Molecular Biology

基　　金：国家自然科学基金（No.81171697）,重庆市自然科学基金（No.2011jjA10047）

摘　　要：目的构建靶向人IRE1a的shRNA干扰质粒（pSUPER-IRE1a）并观察其对人HeLa细胞和HepG2细胞增殖及凋亡的影响。方法设计并合成靶向IRE1a基因的两条shRNA，分别克隆至真核表达载体pSUPER构建重组质粒pS1、pS2，依次转染入HeLa细胞和HepG2细胞中。采用RT—PCR检测pS1、pS2转染前后IRE1a在HeLa细胞和HepG2细胞中的mRNA水平，免疫印迹检测pS1、pS2转染前后IRE1a蛋白的表达；MTT比色法、BrdU／DAPI双免疫荧光法及流式细胞仪分别检测各重组质粒对HeLa细胞和HepG2细胞增殖及凋亡的影响。结果干扰质粒（pSUPER-IRE1a）能有效抑制HeLa细胞和HepG2细胞中IRE1a基因的表达；成功转染后，细胞处于内质网应激（ER stress）状态时，各实验组细胞增殖率及凋亡率与对照组比较，差异均具有统计学意义P〈0．05）。结论成功构建靶向人IRE1a的shRNA真核表达载体pS1、pS2，有效抑制了HeLa细胞和HepG2细胞中IRE1a的表达；细胞处于ERS状态时，IRE1a-shRNA有效促进HeLa、HepG2两种肿瘤细胞的增殖；抑制HeLa和HepG2细胞的凋亡。Objective To construct the targeting IRE1a mRNA interference plasmid pSUPER- IRE1a, study the influence of IRE1a on cell proliferation and apoptosis of HeLa and HepG2 cells during ER Stress. Methods Two shRNA sequences specifically for IRE1a were designed and con- structed into recombinant plasmid pSUPER-IRE1a, then transfected into human HeLa and HepG2 Cells. IRE1 a gene expression levels in HeLa and HepG2 cells were detected with RT-PCR, Western blotting. Cell proliferation was examined by MTT and BrdU/DAPI immunofluorescence assays. Cell apoptosis was determined by flow cytometry. Results The recombinant plasmid pSUPER-IRE1a downregulated IRE1a mRNA and protein levels in HeLa and HepG2 Cells. The cell proliferation rate and cell apoptosis rate were significantly different in experimental groups from control groups （P 〈 0. 05） . Conclusion We successfully constructed the targeting IRE1 a shRNA vector that inhibits mRNA and protein expression of IRE1a in HeLa and HepG2 cells effectively. Besides, IRE1a-shRNA promotes proliferation and inhibits apoptosis of HeLa and HepG2 cells during ER Stress. The IRE1a shRNA vector can be used for further study of IRE1a for its biological functions.

关 键 词：IRE1a SHRNA 细胞增殖 细胞凋亡 

分 类 号：Q78[生物学—分子生物学] Q291