应用细胞工程和转基因技术治疗糖尿病:"胰岛代理细胞"的构建研究

背景由于胰岛细胞分离技术难度较大,目前糖尿病细胞移植治疗尚缺乏理想的细胞来源.目的构建具有生理性胰岛素分泌能力胰岛代理细胞系,为糖尿病基因治疗提供有效手段.设计随机对照的实验研究.地点、材料和干预本研究在第三军医大学附属新桥医院中心实验室进行.以携葡萄糖激酶(glucokinase,GK)基因的逆转录病毒及携人胰岛素原基因突变体(mutant proinsulin gene,MPIN)的逆转录病毒共同感染人肝癌细胞系HepG2细胞,G418筛选,放免法、Western-blotting及RT-PCR鉴定;挑选联合表达GK及成熟胰岛素的HepG2细胞进行葡萄糖反应性胰岛素分泌反应的检测,以单独表达成熟胰岛素的HepG2细胞作对照.主要观察指标不同葡萄糖浓度条件下,联合表达GK及成熟胰岛素的HepG2细胞培养上清中的胰岛素浓度.结果G418筛选3周获阳性细胞克隆,放免法、Western-blotting挑选出两个目的基因表达均较强的HepG2细胞1株,命名为细胞克隆"β";RT-PGR证实细胞"β"中已有两个目的基因的转录;在细胞"β"中获得葡萄糖反应性胰岛素分泌,葡萄糖浓度约1.75～2.00mmol/L时出现胰岛素分泌高峰;而在单独表达成熟胰岛素的HepG2细胞中,各种葡萄糖浓度引起的胰岛素分泌差异无显著性意义(P＞0.05).结论成功构建了具有生理性胰岛素分泌能力的"胰岛代理细胞"系.

Construction of agent islet cell with cell engineering and transgeneic technology on the treatment of diabetes mellitus

BACKGROUND: At present, good cell source for transplant treatment of diabetes is limited, because it is hard to get β cell.OBJECTIVE: To construct"artificial beta cells" which exhibit physiologic glucose-stimulated insulin secretion for effective cure of diabetes.DESIGN: A Randomly controlled trial.SETTING, MATERIAL and INTERVENTIONS: This study was conducted in the center lab of the Third Military Medical University Affiliated Xinqiao Hospital. HepG2 cells were infected with recombinant retrovirus carrying glucokinase(GK)gene and mutant proinsulin(MPIN)gene, then selectively cultured with G418 and identified by radioimmunoassay,SDS-PAGE, Western-blotting and RT-PCR techniques. At last, the dose-response effect of glucose on insulin secretion from HepG2 cells expressed both GK and MPIN gene was tested with HepG2 cells that only expressed MPIN as controls.MAIN OUTCOME MEARSURES: The concentration of insulin was measured in cultivation supernatant of combined expression HepG2 at different glucose levels.RESULTS: HepG2 cells with GK gene and MPIN gene transferred were selectively cultured with G418 and the positive clones obtained in 3 weeks. GK gene and MPIN gene integration and expression were detected by radioimmunoassay, SDS-PAGE, Western-blotting and RT-PCR techniques. The "artificial beta cell" obtained a glucose-stimulated insulin secretion with maximal insulin secretion at 1.75-2.00 mmol/L glucose concentration. While the insulin secretion in HepG2 cell with MPIN gene expression was lack of glucose responsiveness( P ＞ 0.05).CONCLUSION: An artificial beta cell was successfully constructed.