小鼠Reg3α基因cDNA成功转染MIN6细胞株并促其生长_临床医学论文.docVIP

小鼠Reg3α基因cDNA成功转染MIN6细胞株并促其生长_临床医学论文 小鼠Reg3α基因cDNA成功转染MIN6细胞株并促其生长_临床医学论文 作者：崔巍，黄葶，施秉银，刘军 【摘要】 目的 建立一过表达Reg3α cDNA的胰岛MIN6细胞株，研究其在低葡萄糖环境中的生长特性。方法 将全长Reg3α cDNA插入到质粒载体pcDNA3.1中，并将Reg3α和pcDNA3.1空载体用脂质体法转染胰岛MIN6细胞，分别获得Reg3α cDNA过表达和空载体pcDNA3.1MIN6细胞。应用Real和Western blot方法检测这两种细胞在低葡萄糖培养基中Reg3α的表达，并用MTT法测定Reg3α过表达对MIN6细胞生长的影响。结果 有三株Reg3α转染的细胞株显示Reg3α的高表达，在空载体转染的MIN6细胞株中未检测到Reg3α的表达，Reg3α mRNA和蛋白水平分别平均升高6～10倍。用Western blot检测发现Reg3α蛋白释放到培养液。采用MTT法测定，与空载体转染的MIN6细胞株相比，三株Reg3α稳定转染细胞株，7d后细胞数目升高2倍。结论 本研究结果表明已成功地建立了过表达Reg3α cDNA的胰岛MIN6细胞。Reg3α可能具有内分泌作用，促进胰岛细胞生长。 【关键词】 MIN6细胞株；胰岛；Reg基因；转染；生长 ABSTRACT: Objective To create a MIN6 cell line overexpressing murine Reg3α cDNA and to investigate its cell viability character under low glucose concentrations. Methods The fullA was inserted into the plasmid pcDNA3.1(). Then MIN6 cells were transfected with the Reg3αpcDNA3.1 and pcDNA3.1 vector alone to establish Reg3αoverexpression and empty vector MIN6 cell line. Reg3α protein in the low glucose concentration cell medium was detected by Western blot. Cell viability was evaluated by an MTT reduction conversion assay. Results Three Reg3αoverexpression MIN6 cells were confirmed by realtime PCR and Western blot. Reg3α expression was barely detectable in the cells transfected with the empty vector alone. In contrast, the levels of Reg3α mRNA and protein in three pcDNAReg3αtransfected clones were increased by an average 10 and 6fold, respectively. Western blot also revealed Reg3α protein release into the culture medium. In MTT cell proliferation assay, compared to vectortransfection alone, three clones of Reg3αoverexpression MIN6 cells exhibited 2fold increases in cell number over 7 days when cultured in the presence of 10mL/L fetal bovine serum and 5.5mmol/L glucose. Conclusion The Reg3αoverexpression MIN6 cells have been established. Reg3α protein was detectable in culture medium, supporting an endocrine action, and Reg3α overexpression promoted islet cell growth. KEY WORDS: pan