Molecular Biology of B-Cells of Pancreatic Islets of Langerhans : Basic Study to develop Gene Therapy for Diabetes

对胰岛B细胞的分子生物学研究结果如下：1.对一株培养的小鼠胰岛B细胞进行随机cDNA测序，结合Northern印迹分析，获得了约20个胰岛B细胞特异性克隆，获得了从胰岛A细胞分泌免疫抑制因子IL-10或转化生长因子-β的转基因小鼠。两种转化生长因子-β-甘油三酯在NOD小鼠体内尚未表现出糖尿病。在5个胰岛B细胞特异性Th1淋巴细胞中，2个产生干扰素-γ和IL-2，另外3个产生IL-6和10。自身免疫性糖尿病的发生发展需要多种T细胞功能。缺乏CD40的NOD小鼠没有糖尿病，这表明需要CD40来产生自我反应的T细胞。将产生胰岛素原的成纤维细胞移植到HSV-TK加更昔洛韦的失效安全系统中，或过继转移具有识别胰岛抗原能力的IL-10转导的淋巴细胞来建立糖尿病基因治疗的动物模型。由于己糖激酶的转导增加了胰岛素的分泌，而葡萄糖激酶反义mRNA的表达则减少了胰岛素的分泌，胰岛B细胞中的葡萄糖激酶是一种葡萄糖敏感的感受器，在胰岛的内分泌前体细胞中检测到激活素A。胰岛B细胞表达转化生长因子-β。激活素A抑制ATP敏感性钾通道，激活电压依赖性钙通道，导致胰岛素分泌增加。激活素A与Betacelluin协同作用可诱导AR42J的胰腺外分泌细胞分化为胰岛素分泌细胞。

Molecular biological approaches to pancreatic islet B-cells led to following results ;1.Random cDNA sequencing approach comined with Northern blot analysis to a cultured murine cell line of islet B-cells, yielded abount 20 islet B-cell-specific clones.Transgenic mice producing immunosuppressive IL-10 or TGF-beta from pancreatic islet A cells were produced.Transgenic expression of IL-10 in NOD mice aggravated autoimmune diabetes. Two strains of TGF-beta-Tg in NOD mice so far did not exhibit diabetes.Among 5 islet B-cell-specific Th1-lymphoctes, 2 produced IFN-gamma and IL-2, and 3 others produced IL-6 and 10 on top of them. Multiple T-cell functions are necessary for the development of autoimmune diabetes. NOD mice lacking CD40, which is important for T-cell Differentiation, did not become diabetic, suggesting the requirement of CD40 to produce self-reacting T-cells.Transplantaion of proinsulin-producing fibroblasts with the fail-safety systm of HSV-TK plus ganciclovir, or adoptive transfer of IL-10-transduced lymphocytes with the ability to recognize islet-antigens were used to develop animal models of gene therapy for diabetes.Because insulin secretion was increased by transduction of hexokinase, and was decreased by expression of antisense mRNA for glucokinase in transgenic mice, glucokinase in islet B-cells is functioning as a glucose-sensitive sensor.Activin A was detected in the endocrine precursor cells of islets. TGF-beta was detected in pancreatic islet B-cells. Activin A inhibited ATP-sensitive potassium channel, and activated voltage-dependent calcium channels, leading to the increased-insulin secretion. Activin A, in cooperation with betacelluin, induced differentiation of pancreatic exocrine cells of AR42J to insulin-producing cells.