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

朗格汉斯胰岛 B 细胞的分子生物学：开发糖尿病基因疗法的基础研究

• 批准号: 06304034
• 负责人: ITAKURA Mituo
• 金额: $ 8.51万
• 依托单位: The University of Tokushima
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Co-operative Research (A)
• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06304034/
• 关键词: gene therapy; B-cells of pancreatic islets; diabetes mellitus; activin A; TGF-beta; NOD-mice; glucokinase; T細胞; リンパ球

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.

1.随机cDNA测序结合北方印迹分析，获得了20个胰岛B细胞特异性克隆，获得了从胰岛A细胞分泌免疫抑制性IL-10或TGF-β的转基因小鼠，IL-10的转基因表达加重了NOD小鼠自身免疫性糖尿病的发生。NOD小鼠的两株TGF-β-Tg至今未发生糖尿病，5株胰岛B细胞特异性Th 1淋巴细胞中，2株产生IFN-γ和IL-2，3株产生IL-6，10株产生IL-6。多种T细胞功能对于自身免疫性糖尿病的发展是必要的。NOD小鼠缺乏对T细胞分化重要的CD 40，但不发生糖尿病，这表明产生自身反应性T细胞需要CD 40。或过继性转移IL-10转导的淋巴细胞，其具有识别胰岛细胞的能力，抗原用于建立糖尿病基因治疗的动物模型。由于转基因小鼠中胰岛素分泌通过转导己糖激酶而增加，而通过表达葡萄糖激酶的反义mRNA而减少，胰岛B细胞中的葡萄糖激酶作为葡萄糖敏感传感器发挥作用，胰岛内分泌前体细胞中检测到激活素A。在胰岛B细胞中检测到TGF-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β-纤维素共同诱导AR 42 J胰腺外分泌细胞分化为胰岛素分泌细胞。

项目成果

Takashi Yamaoka: "Acute Onset of Diabetic Pathological Changeum Deposit in Diabetic Rat with Human Aldose Reductase cDNA." Diabetologia. (in press). (1995)

Takashi Yamaoka：“用人醛糖还原酶 cDNA 观察糖尿病大鼠急性发生糖尿病病理改变沉积。”

Itakura,M.: "Lessons From Animal Diabetes (Immunostimulatory versus Immunosuppressive Roles of IL-10 in Type I Diabetes)" Smith Gordon (in press), (1996)

Itakura,M.：“动物糖尿病的教训（IL-10 在 I 型糖尿病中的免疫刺激与免疫抑制作用）”Smith Gordon（出版中），（1996 年）

Yamaoka,T.: "Acute Onset of Diabetic Pathological Changes in Transgenic Mice with Human Aldose Reductase cDNA." Diabetologia. 38. 255-261 (1995)

Yamaoka,T.：“使用人醛糖还原酶 cDNA 的转基因小鼠中糖尿病病理变化的急性发作。”

Mitsuo Itakura et al.: ""Immunostimulatory versus Immunosuppressive Roles of IL-10 in Type I Diabetes : Analysis with IL-10-Producing Transgenic Non-Obese Diabetic Mice"" Lessons From Animal Diabetes, Ed.by E.Shafrir. in press.

Mitsuo Itakura 等人：“IL-10 在 I 型糖尿病中的免疫刺激与免疫抑制作用：用产生 IL-10 的转基因非肥胖糖尿病小鼠进行分析”《动物糖尿病教训》，E.Shafrir 编辑。

van Essen: "CD40 Ligand-Transduced Co-Stimulation of T-Cells in the Development of Helper Function." Nature. 378. 620-623 (1995)

van Essen：“CD40 配体转导的 T 细胞共同刺激辅助功能的发展。”