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，胰岛细胞起源于原始导管上皮中的内胚层干细胞，胰岛结构被认为是由胰管细胞出芽形成的，在胚胎18天（E18）的小鼠胚胎中，我们发现了兼具导管细胞和β细胞特征的细胞。通过免疫组织化学，这些细胞对胰岛素（β细胞的标志物）和细胞角蛋白19（导管细胞的标志物）呈阳性。我们假设这些细胞正在分化（3-细胞）。因此，我们试图分离在这些细胞中特异性表达的基因。应用激光捕获显微切割技术和差异显示技术，从导管细胞（细胞角蛋白19阳性细胞）和假定的分化β细胞（细胞角蛋白19阳性细胞和胰岛素阳性细胞）中克隆了在培养的β细胞中表达高于在培养的导管细胞中表达的cDNA，并通过免疫组织化学和原位杂交在胰岛中也表达。我们研究克隆950-5-81，其中一个克隆。克隆950-5-81含有锌指结构域。我们发现该克隆上调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。该克隆将活性激活至2倍。一系列5 '-缺失报告质粒显示锌指反应元件位于-106 B至-55 B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.