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

胰岛素基因治疗系统在糖尿病治疗中的意义：生物人工胰腺B细胞的构建

基本信息

批准号：
06557061
负责人：
SHICHIRI Motoaki
金额：
$ 9.73万
依托单位：
KUMAMOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

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.

本研究将人胰岛素基因转染到CHO细胞、3 T12 -3成纤维细胞、垂体ACTH分泌瘤细胞（AtT 20）和小鼠胰岛素瘤细胞（MIN 6）中。北方印迹分析证实了人胰岛素基因在细胞中的表达。放射免疫分析和高效液相色谱分析显示，转染人胰岛素基因的CHO细胞和3 T12 -3成纤维细胞分泌胰岛素原，而转染人胰岛素基因的AtT 20细胞和MIN 6细胞分泌胰岛素。此外，这些人胰岛素基因转染细胞移植到裸鼠体内后，均能诱导低血糖。转染人胰岛素基因的CHO细胞移植后15 d和转染人胰岛素基因的MIN 6细胞移植后24 h裸鼠死亡，转染人胰岛素基因的MIN 6细胞在培养液中显示出葡萄糖浓度依赖性的胰岛素分泌，而转染人胰岛素基因的AtT 20细胞则无此现象。在灌流实验中，人胰岛素基因转染的MIN 6细胞显示出对葡萄糖有反应的双相胰岛素分泌，而人胰岛素基因转染的AtT 20细胞则没有。另一方面，转染胰岛素基因、葡萄糖转运蛋白2基因和葡萄糖激酶基因的AtT 20细胞在培养基中显示葡萄糖浓度依赖性的胰岛素分泌，虽然在灌流实验中没有发现葡萄糖引起的胰岛素双相分泌，但胰腺B细胞功能基因的构建，在真核细胞中应用生物技术不仅意味着对胰腺B细胞葡萄糖敏感系统、信号转导系统和胰岛素分泌机制的了解，也是胰岛素基因治疗糖尿病的可能模式。