IMPLICATION OF INSULIN GENE-THERAPY SYSTEM IN DIABETES TREATMENT : CONSTRUCTION OF BIO-ARITFICIAL PANCREATIC B CELL

在本研究中，我们将人胰岛素基因导入真核细胞：CHO细胞、3T12-3成纤维细胞、垂体ACTH分泌肿瘤细胞(AtT20)和小鼠胰岛素瘤细胞(MIN6细胞)。经Northern印迹分析证实人胰岛素基因在这些细胞中表达。放射免疫分析和高效液相色谱检测显示，转人胰岛素基因的CHO细胞和3T12-3成纤维细胞分泌胰岛素原进入培养基内，而转人胰岛素基因的AtT20细胞和MIN6细胞则分泌胰岛素原。此外，将这些人胰岛素基因转导的细胞移植到裸鼠体内，均可引起低血糖。裸鼠分别于移植后15天和24小时死亡。转人胰岛素基因的人胰岛素细胞在培养液中表现出葡萄糖浓度依赖性的胰岛素分泌，而转人胰岛素基因的AtT20细胞则不能。在灌流实验中，转人胰岛素基因的MIN6细胞表现出对葡萄糖的双相胰岛素分泌，而转人胰岛素基因的AtT20细胞则没有。而转胰岛素基因、葡萄糖转运蛋白2基因和葡萄糖激酶基因的AtT20细胞在灌流实验中未发现胰岛素对葡萄糖的双相分泌，但在培养上清液中显示了葡萄糖浓度依赖性的胰岛素分泌。在真核细胞中构建胰腺B细胞功能基因生物技术，不仅意味着对胰腺B细胞中未知的葡萄糖传感系统、信号转导系统和胰岛素分泌机制的了解，而且可能是胰岛素基因治疗糖尿病的可能模式。

In this study, we transfected human insulin gene in some eukaryotic cells ; CHO cells, 3T12-3 fibroblasts, pituitary ACTH secreting tumor cells (AtT20) or murine insulinoma cells (MIN6 cells). And their characteristics were evaluated in vitro and in vivo.Expression of human insulin gene in these cells was confirmed by Northern blot analysis. Radioimmunoassay and HPLC revealed that human insulin gene transfected CHO cells and 3T12-3 fibroblasts secreted proinsulin into culture medium, whereas human insulin gene transfected AtT20 cells and MIN6 cells did insulin. In addition, all of these human insulin gene transfected cells induced hypoglycemia, when these cells were transplanted into nude mouse. Nude mouse died at 15 days after the transplantation of insulin gene transfected CHO cells and at 24 hours after the transplantation of insulin gene transfected MIN6 cells.Human insulin gene transfected MIN6 cells showed glucose concentration-dependent insulin secretion in culture media, but human insulin gene transfected AtT20 cells did not. Also, in the perifusion experiment, human insulin gene transfected MIN6 cells revealed biphasic insulin secretion in responce to glucose, but human insulin gene transfected AtT20 cells did not. On the other hand, AtT20 cell transfected with insulin gene, glucose transporter type 2 gene and glucokinase gene showed the glucose concentration-dependent insulin secretion in culture media, although it did not reveal biphasic insulin secretion in responce to glucose in the perifusion experiment.The construction of pancreatic B cell functions gene-biotechnologically in eukaryotic cells might imply not only the understanding of unknown mechanisms of glucose sensing system, signal transduction system and insulin secretion in pancreatic B cell, but also the possible-model of insulin gene-therapy in diabetes treatment.