摘要
目的研究含绿色荧光蛋白(GFP)和人胰岛素原基因的逆转录病毒表达载体在HepG2细胞中的表达。方法将IRES-EGFP片段克隆到含调控元件的人胰岛素原基因逆转录病毒表达载体(pLXSN-GI-Ins)中,构建得到表达质粒pLXSN-GI-Ins-EGFP,经脂质体介导转染HepG2细胞后,各孔分别加入含有不同浓度葡萄糖的培养液继续培养24 h,在荧光显微镜下观察GFP基因的表达,检测细胞上清液中的胰岛素值及考察细胞中胰岛素mRNA的表达量。结果成功构建逆转录病毒表达质粒pLXSN-GI-Ins-EGFP。转染HepG2细胞后48 h,葡萄糖浓度为4.0,12.0,24.0 mmol/L,表达GFP基因的细胞数目占总细胞数目的比值分别为(4.00±0.15)%,(10.00±0.07)%,(25.00±0.10)%;胰岛素分泌量分别为(4.05±0.82),(8.67±0.62),(30.70±1.55)mU/L,不同葡萄糖浓度诱导的胰岛素mRNA表达量明显不同。结论含GFP和调控元件的人胰岛素原基因逆转录病毒表达载体能够在HepG2细胞中成功表达,并且GFP基因的表达量反映了人胰岛素的分泌量。
Objective To study the expression of the retroviral expression vector carrying green fluorescent protein gene（GFP） and human insulin gene in HepG2 cell. Methods The fragment encoding IRES-EGFP was cloned into the retroviral expression vector carrying human insulin gene with regulatory element（pLXSN-GI-Ins）. The expression plasmid of pLXSN-GI-Ins-EGFP were constructed. The plasmid was transfected into HepG2 cells by using lipofectamine. To observe the expression of GFP gene by fluorescence microscope and determine insulin value in clear supernatant liquid of cells cultured in medium with different glucose concentrations for 24 h. RT-PCR was used to determine the expression levels of recombinant insulin mRNA at three glucose concentrations. Results The retroviral expression plasmid of pLXSN-GI-Ins-EGFP was successfully constructed. At the glucose concentration of 4.00, 12.00 and 24.00 mmol/L, the expression of GFP gene was （4.00±0.15） %, （10.00±0.07） %, （25.00±-0.10） %. The insulin secretion was （4.05±0.82）, （8.67±0.62） and （30.70±1.55） mU/L. The mRNA expression of insulin gene were positively related to different glucose concentration. Conclusion The retroviral expression vector carrying green fluorescent protein gene（GFP） and human insulin gene with regulatory element was successfully expressed in HepG2 cells, and the insulin secretion was determinated by the expression of GFP gene.
出处
《福建医科大学学报》
2007年第5期393-395,399,共4页
Journal of Fujian Medical University
基金
福建省教育厅科研基金资助项目(JB05020)
