期刊文献+

RegIα cDNA过表达质粒的构建及对胃癌细胞增殖凋亡的影响 被引量:1

Construction of RegIα over-expression vector and its effect on proliferation and apoptosis of gastric cancer cells
下载PDF
导出
摘要 目的:构建再生蛋白Iα(regeneration gene Iα,RegIα)的cDNA过表达质粒表达载体,在体外评价其对胃癌细胞株增殖及其凋亡的影响。方法:根据RegIαcDNA的全序列设计引物,用RT-PCR的方法从胃黏膜组织的总RNA中获取RegIα的cDNA编码区域的全序列,经测序其与目的基因序列一致。将获取的RegIα序列经TA克隆和亚克隆至pIRES2-EGFP真核表达质粒载体中;将构建的RegIα过表达质粒载体pIRES2-RegIα-EGFP转染胃癌细胞株MKN28,并用空载体pIRES2-EGFP作为对照,利用G418筛选稳定表达pIRES2-RegIα-EGFP的MKN28细胞株。通过RT-PCR及Western blot检测转染后细胞RegIα在mRNA及蛋白质水平的表达;MTT法和流式细胞仪分别检测RegIα过表达后MKN28细胞增殖及拮抗H2O2诱导凋亡的变化。结果:成功构建RegIαcDNA的全序列表达质粒。RT-PCR及Western blot检测显示:转染pIRES2-EGFP质粒的MKN28细胞RegIαmRNA及蛋白表达水平明显升高(P<0.05);MTT试验显示:转染后细胞增殖能力明显升高(P<0.05),而且拮抗H2O2凋亡的能力增强(P<0.05)。结论:RegIα的过表达能明显增强胃癌细胞MKN28的增殖和拮抗H2O2诱导凋亡的能力。 Objective: To construct RegIα over-expression vector and to evaluate the effect of RegIα on the proliferation and apoptosis of gastric cancer MKN28 cells in vitro.Methods: Full sequence of RegIα cDNA was amplified from normal gastric tissue samples by RT-PCR and cloned into pIRES2-EGFP vector.RT-PCR and Western blot were performed to detect expression levels of RegIα in MKN28 cells.The effects of over-expression RegIα on cell proliferation was measured by MTT assay and apoptosis was detected by flow cytometry.Results: RegIα cDNA over-expression vector of pIRES2-RegIα-EGFP was successfully constructed.The expressions of RegIα in MKN28 cells,including mRNA and protein levels,were significantly increased after stable transfection,which resulted in cell proliferation and anti-apoptotic effect induced by H2O2.Conclusion: The over-expression of RegIα can promote cell proliferation and reduce cell apoptosis when induced by H2O2 in gastric cancer cells.
出处 《浙江大学学报(医学版)》 CAS CSCD 北大核心 2010年第5期499-505,共7页 Journal of Zhejiang University(Medical Sciences)
基金 浙江省自然科学基金(Y206280) 浙江省教育厅科研项目(Y20070071)
关键词 胃肿瘤 再生/遗传学 细胞系 肿瘤 逆转录聚合酶链反应 遗传载体 质粒 细胞凋亡 细胞增殖 转染 Stomach neoplasms Regeneration/gene Cell line tumor Reverse transcriptase polymerase chain reaction Genetic vectors Plasmids Apoptosis Cell proliferation Transfection
  • 相关文献

参考文献2

二级参考文献4

共引文献21

同被引文献17

  • 1Yoshikazu Kinoshita,Shunji Ishihara,Yasunori Kadowaki,Hiroshi Fukui,Tsutomu Chiba.Reg protein is a unique growth factor of gastric mucosal cells[J]. Journal of Gastroenterology . 2004 (6)
  • 2RIVERA F,VEGA-VILLEGAS M E,LOPEZ-BREAM F.Chemotherapy of advanced gastric cancer.. Cancer Treatment Reviews . 2007
  • 3KINOSHITA Y,ISHIHARA S,MIYAOKA Y,etal.Reg protein is overexpressed in gastric cancercells,where it activates a signal transductionpathway that converges on ERK1/2 to stimulategrowth. FEBS Letters . 2002
  • 4Sekikawa,A,Fukui,H,Fujii,S.REG Iα protein may function as a trophic and/or anti-apoptotic factor in the development of gastric cancer. Gastroenterology . 2005
  • 5Sekikawa A,Fukui H,Fujii S.RegⅠα protein mediates an anti-apoptotic effect of STAT3 signaling in gastric cancer cells. Carcinogenesis . 2008
  • 6Fukui H,Fujii S,Takeda J,et al.Expression of RegⅠ alpha protein in human gastric cancers. Digestion . 2004
  • 7DharDK,Udagawa J,Ishihara S,et al.Exp ression of regenerating gene I in gastric adenocarcinomas: correlation with tumor differentiation status and patient survival. Cancer . 2004
  • 8Yonemura Y,Sakurai S,Yamamoto H,et al.REG gene expression is associated with the infiltrating growth of gastric carcinoma. Cancer . 2003
  • 9Vickers T A,Koo S,Bennett C F,et al.Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. Journal of Biological Chemistry . 2003
  • 10Elbashir SM,Harborth J,Weber K,et al.Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods . 2002

引证文献1

二级引证文献1

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部