Molecular Analysis of a Glucose-concentration sensing system which Regulates Insulin Gene Expression

从三方面研究了体细胞基因治疗中胰岛素基因表达的调控原理。第一种方法通过重组技术利用分泌人胰岛素原的成纤维细胞。用编码小鼠基因组CD8.2基因的重组质粒转染这些纤维玻璃细胞。这些双重转染的细胞被腹腔移植到链脲佐菌素诱导的糖尿病C3H小鼠。移植后第30天，血糖浓度从治疗前的430 mg/dl显著下降到80 mg/dl。为了清除移植细胞，给药抗cd8.2单克隆抗体，使血糖水平恢复到治疗前水平，证明移植细胞完全清除。第二种方法使用的MIN-6细胞来源于大鼠胰岛素启动子- t抗原转基因小鼠。将该细胞系移植到糖尿病小鼠C57BL06后，血糖水平立即恢复到正常范围。葡萄糖耐量试验显示，随着移植后时间的延长，移植细胞的葡萄糖反应性降低。第三，通过不同葡萄糖浓度培养该b细胞株，研究了胰岛素分泌的葡萄糖依赖机制，发现胰岛素mRNA的变化很小，而胰岛素分泌却有6倍的差异。这表明翻译控制比转录控制更重要。今后对胰岛素分泌的葡萄糖依赖性调控的研究应着眼于对这种MIN-6细胞系葡萄糖依赖性的分子分析。利用b细胞衍生的细胞系和成纤维细胞作为体细胞基因治疗糖尿病的可能靶点也很重要。

The principle of controling insulin gene expression in somatic cell gene therapy was studied in three ways. The first method used fibroblasts which secrete human proinsulin by recombinant technology. these fibroglasts were transfected with the second recombinant plasmid encoding mouse genomic CD8.2 gene. These doubly transfected cells were trans-planted intraperitoneally to streptozocin-induced diabetic C3H mice. The blood glucose concentrations were remarkably decreased from 430 mg/dl of the pre-treatment level to 80 mg/dl at the 30th day after the transplantation. To remove the transplanted cells, anti-CD8.2 monoclonal antibody was administered, which lead to the reversal of blood glucose level to the pre-treatment level, proving the complete removal of transplanted cells. The second method used MIN-6 cells which originated from transgenic mice with a Rat-Insulin-Promoter-T-antigen transgene. The transplantation of this cell line to diabetic C57BL06 mice immediately improved blood glucose levels to the normal range. Along with the time after transplantation, glucose responsiveness of transplanted cells decreased as estimated by glucose tolerance test. Thirdly glucose-dependent mechanism of insulin secretion in this B-cell line was examined by culturing this cell line in different glucose concentrations, which showed minimul change of insulin mRNA in contrast to 6-fold difference of insulin secretion. This suggests the importance of translational control rather than transcriptional control. The future study on the glucose-dependent regulation of insulin secretion should be directed to the molecular analysis of glucose-dependency in this MIN-6 cell line. It is also important to use B-cell-derived cell lines as well as fibroblasts as the possible target of somatic cell gene therapy against diabetes.