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

调节胰岛素基因表达的葡萄糖浓度传感系统的分子分析

基本信息

批准号：
03454518
负责人：
ITAKURA Mitsuo
金额：
$ 4.16万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03454518/
关键词：
gene therapy B-cells of pancreatic islets diabetes mellitus fibroblasts proinsulin glucose-dependency insulin gene

项目摘要

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.

从三个方面探讨了体细胞基因治疗中胰岛素基因表达调控的原理。第一种方法使用通过重组技术分泌人胰岛素原的成纤维细胞。用编码小鼠基因组CD8.2基因的第二重组质粒转染这些成纤维细胞。将这些双重转染的细胞腹膜内移植到链脲佐菌素诱导的糖尿病C3 H小鼠中。移植后第30天血糖浓度从治疗前的430 mg/dl降至80 mg/dl。为了去除移植的细胞，给予抗CD8.2单克隆抗体，导致血糖水平逆转至治疗前水平，证明移植的细胞已完全去除。第二种方法使用MIN-6细胞，其来源于具有大鼠胰岛素启动子T抗原转基因的转基因小鼠。将该细胞系移植到糖尿病C57 BL 06小鼠后，血糖水平立即改善至正常范围。糖耐量试验显示，沿着移植后时间的延长，移植细胞的葡萄糖反应性逐渐降低。第三，通过在不同的葡萄糖浓度下培养该细胞系来检测该细胞系中胰岛素分泌的葡萄糖依赖性机制，其显示胰岛素mRNA的变化最小，而胰岛素分泌的差异为6倍。这表明翻译控制的重要性，而不是转录控制。胰岛素分泌的葡萄糖依赖性调节的未来研究应致力于葡萄糖依赖性的分子分析在这个MIN-6细胞系。同样重要的是使用B细胞来源的细胞系以及成纤维细胞作为体细胞基因治疗糖尿病的可能靶点。