人胰岛素原基因在体外培养的NIH3T3细胞中的基因转移和表达的研究

Gene transfer and expression of human proinsulin gene in NIH 3T3 cells in vitro

目的 研究人胰岛素原基因在体外培养的小鼠成纤维细胞NIH 3T3细胞系的基因转移和表达情况 ,进而为 1型糖尿病的基因治疗提供实验依据。方法 采用脂质体转染法分别将未携带外源基因的反转录病毒载体 pLNCX和携带大鼠磷酸烯醇式丙酮酸羧激酶 (PEPCK)启动子 人胰岛素原基因 (INS)的重组反转录病毒载体pN PEPCK INS转染到体外培养的NIH 3T3细胞系中。转染后 72小时 ,用G418筛选出阳性细胞克隆并培养至 2 4天 ,提取细胞的染色体DNA ,行PCR扩增 ,电泳检查转入基因情况。同时用放射免疫分析方法监测培养液的胰岛素水平及转染后 2 4天的细胞内胰岛素水平。结果 PCR证实pLNCX和 pN PEPCK INS均整合到NIH 3T3细胞的染色体中。培养液的胰岛素水平在转染前后均无明显变化 (P >0 .0 5 ) ,而转入 pN PEPCK INS的NIH 3T3细胞内的胰岛素水平则于转染后 2 4天明显高于未转染组 (P <0 .0 1)及pLNCX转染组 (P <0 .0 5 )。结论人胰岛素原基因在NIH 3T3细胞系的转移成功和在细胞内的高效表达说明基因治疗有望成为 1型糖尿病的有效治疗途径之一。

Objective To study human proinsulin gene transfer and expression in NIH 3T3 fibroblast cell line. Methods Retroviral vectors pLNCX and recombinant pN PEPCK INS were transfected seperately into NIH 3T3 cell by liposome mediated transfection. The transfected cells were grown in DMEM medium containing G418 at 72 hours after transfection, and the clones of cells were selected at the 7th day and continued to grow in G418 medium until the 24th day. Chromosomal DNA was isolated from transfected NIH 3T3 cells and polymerase chain reaction (PCR) was performed. PCR products were analyzed by electrophoresis and insulin levels were monitored by radioimmunoassay. Results PCR products proved that both pLNCX and pN PEPCK INS were integrated into the chromosome of NIH 3T3 cells. There were no changes in extracellular insulin levels during the 24 days after transfection (P>0.05). However, intracellular insulin levels at the 24th day after transfection increased significantlyascomparedwithuntransfectedgroup (P<0.01) and pLNCX transfected group (P<0.05). Conclusion Human proinsulin gene was transfected successfully and expressed efficiently in NIH 3T3 cells, indicating that the gene therapy of type 1 diabetes would be possible.