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