Gene Therapy for diabetes mellitus with non-endocrine cells

非内分泌细胞糖尿病基因治疗

• 批准号: 11671137
• 负责人: SASAKI Takashi
• 金额: $ 2.11万
• 依托单位: Jikei University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671137/
• 关键词: Celltherapy; Insulin; Tissue Engineering; Pancreas; Differentiation; Stem cell; Precursor; Mesenchyme; プロホルモン; プロセッシング; フューリン; 膵島移植; 1型糖尿病; 再生医学; レトロウイルス; レトロウィルスベクター; GFP; 細胞内輸送; レトロウイルスベクター; 前脂肪細胞

We have focused on generation of non-endocrine cell lines that could secret mature human insulin. If human mature insulin would be generated in an ectopic cell, or a non-endocrine cell, biosynthesized proinsulin Should be processed. For this purpose, nucleotide sequences of C-peptide domain at the BC and CA junctions of human insulin CDNA were changed for the prohormone convertase, furin, recognition site. Murine mesenchymal progenitor celllines including LI preadipocytes and C2C12 myoblasts were then engineered by the modified CDNA with a recombinant retroviral vector.Human insulin was detected by a human-specific immunoassay in culture media of the engineered cells, and the cells could be stained immunocytochemically by specificanti-human insulin antibody. Biochemical analysis using reversephase HPLC and mass spectrometry (MALDI) revealed that the insulin secreted from the transformed cell lines was identical to native human insulin. When the cells were transplanted to diabetic mice with immunoisolating chamber consisted of semipermeable membrane for evaluation of biological activity of secreted insulin, blood glucose level of the mice were recovered to near normal range while that of mice with non-engineered cells showed no change, demonstrating the biological potency of transplantation of the engineered cells. In the observation of insulin secretion from the engineered precursor cells, insulin secretion rate is increased according to the differentijation of the cells would proceeds. This finding should be extremely important for surrogate cell-based therapy in general. Our present study suggests that the novel technologies could enable surrogate cell therapy for severe form of human diabetes mellitus.

我们一直专注于产生能够分泌成熟人类胰岛素的非内分泌细胞系。如果人类成熟的胰岛素将在异位细胞或非内分泌细胞中产生，则应该处理生物合成的胰岛素原。为此，在人胰岛素CDNA的BC和CA连接处的C-肽域的核苷酸序列被改变，以识别前激素转换酶、呋喃，识别位点。将修饰后的CDNA与逆转录病毒载体共同构建成肌细胞系LI前体脂肪细胞和C2C12成肌细胞，用人胰岛素免疫分析法检测细胞培养上清中的人胰岛素，并用特异性抗人胰岛素抗体对细胞进行免疫细胞化学染色。用反相高效液相色谱-质谱仪(MALDI)进行生化分析表明，转化细胞株分泌的胰岛素与天然人胰岛素相同。用半透膜构成的免疫隔离室将细胞移植到糖尿病小鼠体内，评价其分泌胰岛素的生物活性，小鼠的血糖水平恢复到接近正常范围，而非工程细胞的小鼠血糖水平无变化，显示了工程细胞移植的生物学效力。在对工程前体细胞胰岛素分泌的观察中，胰岛素的分泌速率随着细胞分化的进行而增加。这一发现对于总体上基于替代细胞的治疗应该是极其重要的。我们目前的研究表明，这些新技术可以使替代细胞治疗严重的人类糖尿病成为可能。

项目成果

Kei Fujimoto, et al.: "Piccolo, a Ca^<2+> Sensor in pancreatic β-Cells"The Journal of Biological Chemistry. 277・52. 50497-50502 (2002)

Kei Fujimoto等人：“Piccolo，胰腺β细胞中的Ca^2+传感器”生物化学杂志277・52（2002）。

Yamamoto, Junko, et al.: "PPARγ2 Pro12Ala polymorphism and insulin resistance in Japanese hypertensive patients"Hypertens Res. 25(1). 25-29 (2002)

Yamamoto, Junko, et al.：“PPARγ2 Pro12Ala 多态性和日本高血压患者的胰岛素抵抗”Hypertens Res 25(1) (2002)。

Takashi Sasaki, et al.: "Gene and cell-based therapy for diabetes mellitus"Endocr Pathol, summer issue. in press. (2003)

Takashi Sasaki 等人：“糖尿病的基因和细胞疗法”Endocr Pathol，夏季期。

Koichiro Yamasaki: "Differentiation-induced insulin secretion from nonendocrine cells with engineered human proinsulin cDNA"Biochem Biophys Res Commn. 265. 361-365 (1999)

Koichiro Yamasaki：“用工程化的人胰岛素原 cDNA 分化诱导非内分泌细胞分泌胰岛素”Biochem Biophys Res Commn。

Nemoto, Masami, et al.: "Differential effect of PPAEγ2 variants in the development type 2 diabetes between Japanese and Japanese Americans"Diab Res Clin Pract. 57. 131-137 (2002)

Nemoto，Masami 等：“PPAEγ2 变体在日本人和日裔美国人之间发展 2 型糖尿病中的差异效应”Diab Res Clin Pract. 57. 131-137 (2002)。