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

为了定量胰岛β细胞质量，我们试图开发一种无创且易于测量的系统。我们设计了一种β细胞模型，其组成性分泌人工报告分子，反映β细胞质量，用常规方法测量。首先，我们构建了三种类型的人工融合蛋白。一种是将人白蛋白信号序列与人C肽融合（Alb-hc），另两种是将小鼠IgK信号序列与人C肽cDNA融合（hCPR串联），另一种是同时含有YFP cDNA（hCPR/YFP）。我们已经确定了这些蛋白质在培养细胞系（Cos 7，HEK 293）外的表达和分泌，这意味着这些信号肽在体内起分泌信号的作用。除此之外，我们还可以用人C肽ELISA试剂盒对这些融合蛋白进行检测，这些报告分子显然很容易被检测到，作为进一步的研究，我们将这些报告分子导入小鼠β细胞系MIN 6细胞中，并证明它们在其中的表达和分泌。重要的是要确认这些报告基因分泌是否是组成型的。我们必须确定这些报告基因分泌的改变与几种条件（葡萄糖刺激等）无关。这样我们就可以用这些报告基因来计算β细胞的数量。在建立了导入这些融合基因的转基因小鼠后，我们正在制造这些小鼠和其他糖尿病小鼠的杂交小鼠。这些杂交小鼠将证明β细胞群的改变，直到糖尿病发作。沿着糖尿病发病或进展机制的阐明，这种无创性β细胞质量定量系统将有助于开发抑制β细胞质量减少的新药或促进β细胞再生治疗。

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.