Identification of genes that are involved in differentiation of pancreatic ductal cells to pancreatic β-cells.

在发育中的内分泌胰腺中，胰岛细胞由位于原始管上皮内的内胚层干细胞产生，胰岛结构被认为是由胰管细胞出芽形成的。在胚胎第18天的小鼠胎儿（E18）中，我们发现细胞同时具有导管细胞和β细胞的特征。这些细胞免疫组化检测胰岛素（β细胞的标志物）和细胞角蛋白19（导管细胞的标志物）阳性。我们假设这些细胞正在分化（3个细胞）。因此，我们试图分离出在这些细胞中特异性表达的基因。利用激光捕获显微解剖技术和差异显示方法，从导管细胞（细胞角蛋白19阳性细胞）和可能分化的3细胞（细胞角蛋白19和胰岛素阳性细胞）中克隆出在培养的β细胞中比在培养的导管细胞中表达更多的cdna，并通过免疫组织化学或原位杂交在胰岛中表达。我们研究了克隆950-5-81，其中一个克隆。克隆950-5-81含有锌指结构域。我们发现克隆上调了Glut - 2基因的表达。然后，我们通过荧光素酶分析检测了克隆对Glut 2启动子活性的影响。克隆激活了2倍的活性。一系列的5'缺失报告质粒表明，锌指响应元件位于106b - 55b区域。

In the developing endocrine pancreas, islet cells arise from endodermal stem cells which lie in the primitive duct epithelium, and the islet structure is thought to be formed by budding from the pancreatic duct cells.In mouse fetus of embryonic day 18 (E18), we found cells that have both characters of ductal cells and of β-cells. Those cells were positive for insulin (a marker of β-cells) and cytokeratin 19 (a marker of ductal cells) by immunohistochemistry. We assumed that those cells are differentiating (3-cells. We, therefore, tried to isolate genes that are specifically expressed in those cells. Using laser capture microdissection technique and differential display method from the ductal cells (cytokeratin 19-positive cells) and the putative differentiating (3-cells (both cytokeratin 19-and insulin-positive cells), we cloned cDNAs that are expressed more in the cultured β-cells than in the cultured ductal cells by Northern blot and are also express in the pancreatic islet by immunohistochemistry or in situ hybridization. We study the clone 950-5-81, one of those clones. The clone 950-5-81 contains a zinc finger domain. We found that the clone upregulates Glut 2 gene expression. We then examined the effect of the clone on Glut 2 promoter activity by luciferase assya. The clone activated the activity to 2-fold. A series of 5'-deletion reporter plasmids revealed that the Zn finger responsive element reside in a region of-106 b to-55 b.