The Development of the Model Mice Having the System that Easily Measuring their Pancreatic Islet Beta Cell Mass

具有可轻松测量胰岛β细胞质量的系统的模型小鼠的开发

基本信息

批准号：
15590941
负责人：
MATSUBARA Atsushi
金额：
$ 2.24万
依托单位：
YAMAGUCHI UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

To quantify the islet beta cell mass, we tried to develop the noninvasive and easily measurable system. We designed a beta cell model, which constitutively secrete an artificial reporter molecule, reflecting the beta cell mass, to be measured with conventional methods. First of all, we have constructed three types of artificial fusion proteins. One has the human albumin signal sequence with the human C-peptide (Alb-hc), another two types of constructs both have the mice IgK signal sequence fused with human C-peptide cDNA (hCPR tandem) while the one has also YFP cDNA (hCPR/YFP). We have determined those proteins expression and secretion outside of cultured cell lines (Cos7, HEK293), meaning these signal peptides play the role as the secreting signal in vivo. Besides we could detect and measure those fusion proteins with the human C-peptide ELISA kit, these reporter molecules are apparently easy to be measured.As the further investigation, we will import those reporters into MIN6 cells, mouse beta cell line, and prove those expression in it and secretion. It is important to confirm whether those reporters secretion is constitutive or not. We have to determine the alteration of those reporters secretion independent of several conditions (glucose stimulation etc.) so that we will be able to use these reporters to calculate beta cell mass.After establishing transgenic mice imported those fusion genes, we are making hybrid mice between these mice and other diabetic mice. These hybrid mice will demonstrate the alteration of beta cell mass till the onset of diabetes mellitus. Along with the elucidation of the mechanism of the diabetes onset or progression, this noninvasive beta cell mass quantifying system will contribute to developing the new drugs to inhibit beta cell mass reduction or to the advancing of beta cell regenerative therapy.

为了量化胰岛β细胞质量，我们试图开发非侵入性且易于测量的系统。我们设计了一个Beta细胞模型，该模型将用常规方法测量以反映β细胞质量的人工记者分子分泌。首先，我们构建了三种类型的人工融合蛋白。一个具有人类白蛋白信号序列，具有人类C肽（ALB-HC），另一种类型的构建体都具有与人C肽cDNA（HCPR串联）融合的小鼠IGK信号序列，而一个构建体也具有YFP cDNA（HCPR/YFP）。我们已经确定了在培养的细胞系以外（COS7，HEK293）之外的那些蛋白质表达和分泌，这意味着这些信号肽在体内发挥了作用。此外，我们可以检测并测量与人C肽ELISA试剂盒的融合蛋白，这些报告基因显然很容易被测量。随着进一步的研究，我们将这些记者将这些记者进口到MIN6细胞，小鼠Beta细胞系，并证明其在IT中的表达和分泌。重要的是要确认这些记者分泌是否是本构的。我们必须确定这些记者分泌的改变，与几种条件（葡萄糖刺激等）无关，以便我们能够使用这些记者来计算β细胞量。建立转基因小鼠进口这些融合基因后，我们正在使这些小鼠之间的杂化小鼠和其他糖尿病小鼠之间进行混合。这些杂种小鼠将证明β细胞质量的改变，直到糖尿病发作。除了阐明糖尿病发作或进展的机制外，这种非侵入性β细胞量量化系统将有助于开发新药物以抑制β细胞质量的减少或β细胞再生疗法的前进。