人胰岛素基因在NIH3T3细胞中的基因转染和表达

目的　研究人胰岛素基因在体外培养的小鼠成纤维细胞系的基因转染和表达。方法　采用壳聚糖转染法将重组的人胰岛素基因表达质粒转染鼠成纤维细胞 (NIH3T3) ,转染后 72h ,经G4 18抗性筛选出阳性克隆并培养到第 2 4d ,用免疫组织化学方法检测胰岛素的表达 ,同时用ELASA法监测转染后 2 4d的细胞培养液的胰岛素水平。结果　转染PCMV .INS的NIH3T3细胞培养液的胰岛素水平明显高于未转染组 (P <0 .0 1)及PCMV转染组 (P <0 .0 1) ,未转染组与PCMV转染组细胞培养液的胰岛素水平无明显变化 (P >0 .0 5 )。结论人胰岛素基因在NIH3T3细胞系的转染成功和表达说明基因治疗有望成为 1型糖尿病的重要治疗手段 ,壳聚糖是一种很有前途的胰岛素基因载体。

Gene Transfection and Expression of Human Insulin Gene in Fibroblasts in Vitro

Objective To study human insulin gene transfection and expression in NIH3T3 cell by chitosan-mediated transfection. Methods An expression vector for human insulin gene was constructed by recombinant gene technique and introduced into NIH3T3 cells by chitosan-mediated DNA transfection. The transfection cells were grown in DMEM medium containing G418 at 72 hours after transfection,and the clones of cells were selected and continued to grow in G418 medium until the 24th day.The transfected cells by immunohistochemical and insulin levels were monitored by ELASA.Results Extracellular insulin levels in PCMV.INS transfected group increased significantly as compared with that in untransfected group(P<0.01) and PCMV transfected group(P<0.01),while there were no changes of extracellular insulin levels in untransfected group and PCMV group(P>0.05).Conclusion Human insulin gene was transfected successfully by chitosan-mediated and expressed efficiently in NIH3T3 cells,indicating chitosan would be a promising non-viral vector and the gene therapy of type 1 diabetes would be possible.