摘要
目的 构建血红素氧化酶-1（HO-1）基因的真核过表达载体,观察转染人结肠癌细胞株Caco-2细胞后对其生长的影响和细胞定位.方法 构建人pShuttle-CMV-HO1载体,经测序、酶切鉴定正确后,采用脂质体转染法将载体转入Caco-2细胞中,经G418筛选并建立稳定过表达Caco-2细胞HO-1基因的细胞株.实验分为2组,分别为实验组（Caco-2-pS/HO-1）、对照组（Caco-2-pS）,每组设3个复孔,应用荧光定量聚合酶链反应（FQ-PCR）法和Western blot法分别检测HO-1 mRNA和蛋白的表达水平；通过免疫荧光法观察HO-1分子在细胞内的分布；采用细胞计数试剂盒（CCK-8）法检测HO-1基因过表达后对Caco-2细胞的增殖影响.结果 酶切及测序显示pShuttle-CMV-HO1载体构建符合预期.与Caco-2-pS组比较,Caco-2-pS/HO-1组细胞HO-1基因mRNA表达明显增加（63.55±0.86比3.66±0.36,P＜0.05）,HO-1蛋白表达呈现相同趋势；对转染目的载体的细胞应用m-CY3标记的抗体进行免疫荧光实验,激光共聚焦显微镜观察到HO-1分子主要分布于细胞质与细胞膜；Caco-2-pS/HO-1组较Caco-2-pS组细胞增殖数量明显增加[（5.43±0.24）个比（3.25±0.25）个,P＜0.05].结论 成功构建HO-1基因真核过表达载体,HO-1蛋白主要表达于细胞膜及细胞质中,HO-1蛋白异常表达可以促进Caco-2细胞增殖,从而对体外肠黏膜屏障模型起到保护作用.
Objective To investigate the potential role and localization of heme oxygenase-1 （HO-1） gene in human colorectal cancer Caco-2 cell line by constructing the overexpression vector of HO-1 gene.Methods A overexpression vector of HO-1 gene was constructed and transfected into Caco-2 cells by lipofectin-mediated method after the identification of sequencing and enzyme digestion.Cells （Caco-2-pS/HO-1）stably overexpressing HO-1 gene were selected by using G418.Cells were divided into two groups with three repetitive holes in each group.Caco-2 cells untransfected were used as controls.The expression levels of HO-1 gene were detected by using real-time fluorescent quantitative polymerase chain reaction （FQ-PCR）and Western blotting,the cellular localization of HO-1 in Caco-2 cells was observed by cell immunofluorescence and the biological function of HO-1 gene was analyzed by cell counting kit-8 （CCK-8）.Results The result of sequencing and enzyme digestion showed the construction of overexpression vector of HO-1 gene in line with the expected.As compared with Caco-2-pS cells,the expression of ZO-1 mRNA in Caco-2-pS/HO-1 cells was markedly increased （63.55 ± 0.86 vs.3.66 ± 0.36,P 〈 0.05）,and HO-1 protein had the same tendency.In transfected Caco-2 cells in immunofluorescence with C-Y3 labeled antibody under confocal laser microscope,it was found HO-1 molecules were mainly distributed in the cytoplasm and cell membrane.The cell growth activity showed same tendency in Caco-2-pS/HO-1 group in comparison to Cacl-2-pS group （5.4300 ±0.2497 vs.3.2500 ±0.2477,P〈0.05）.Conclusion The overexpression vector of HO-1 gene was constructed successfully and the high expression of HO-1 located in cytomembrane and cytoplasm could promote the proliferation of Caco-2 cells,which could protect intestinal mucosa in thein vitro model.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2013年第11期2318-2320,共3页
Chinese Journal of Experimental Surgery
基金
山东省科技发展计划资助项目（2011GSF11846）
