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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 the gene transfer and expression of human proinsulin gene in NIH 3T3 cell line cultured in vitro and to provide an experimental basis for the gene therapy of type 1 diabetes mellitus. Methods Recombinant retroviral vector pLNCX without exogenous gene and human proinsulin gene (INS) carrying rat phosphoenolpyruvate carboxykinase (PEPCK) promoter were reverse transcribed by lipofectamine The viral vector pN PEPCK INS was transfected into in vitro cultured NIH 3T3 cell line. 72 hours after transfection, positive clones were screened by G418 and cultured for 24 days. The chromosomal DNA of the cells was extracted and subjected to PCR amplification and electrophoresis to check the gene transfection. At the same time, the insulin level of the culture medium and the intracellular insulin level 24 days after transfection were monitored by radioimmunoassay. Results PCR confirmed that both pLNCX and pN PEPCK INS were integrated into the chromosome of NIH 3T3 cells. The level of insulin in culture medium did not change significantly before and after transfection (P> 0.05), while the level of insulin in NIH 3T3 cells transfected with pNPEPCK INS was significantly higher than that of non-transfected cells Group (P <0.01) and pLNCX transfection group (P <0.05). Conclusions The successful transfer of human proinsulin gene in NIH 3T3 cell line and the high expression in cells indicate that gene therapy is expected to be an effective treatment for type 1 diabetes mellitus.