Analysis of β cell differentiation in diabetic pancreas induced by betacellulin with special reference to the role of Pax family gene

在内分泌胰腺的发育过程中，各种转录因子参与调节生长和分化因子的表达。其中，Pax家族在胰岛素分泌β细胞的终末分化过程中起着重要作用。另一方面，在β细胞耗竭的糖尿病胰腺中发生新生的机制还知之甚少。因此，我们研究了选择性四氧嘧啶灌流诱导的葡萄糖耐受小鼠胰腺中包括Pax家族在内的转录因子的表达对β细胞新生过程的影响。我们还试图利用AR42J细胞中引入的Pax6和Pax4基因的显性负性形式来阐明这些基因的作用。胰岛样细胞团与胰管细胞衬里密切相关，免疫组织化学分析显示，Pax1、Islet1、Nkx2.2和Pax6等转录因子在部分胰管细胞和ICC细胞中表达，提示Pax6参与了糖尿病胰腺β细胞的新生，其再生(新生)过程与胎儿内分泌细胞发育过程相似。然而，在发育过程中对β细胞的最终分化起重要作用的Pax4水平是检测不到的。β细胞蛋白是EGF家族的生长因子之一，可以诱导AR42J细胞分化为胰岛素分泌细胞，增加ICCs的数量，改善糖耐量异常。为了阐明Pax4和Pax6在β细胞分化中的作用，我们试图在AR42J细胞中获得这些基因的显性阴性形式的稳定转化子，但这是困难的，而且我们不能用体外实验系统来分析这两个Pax基因在β细胞分化中的作用。

In the development of endocrine pancreas, various transcription factors that regulate the expression of growth and differentiation factors are involved. Among them, Pax family is known to have important role in the final differentiation of insulin-secreting β cells. On the other hand, little is known in the mechanism of neogenesis which occurs in the β cell depleted diabetic pancreas. We thus studied the process of β cell neogenesis with special reference to the expression of transcription factors including Pax family in pancreas of glucose intolerant mice induced by selective alloxan perfusion. We also tried to clarify the role of Pax 6 and 4 using dominant negative form of these genes introduced in AR42J cells. ICCs(Islet-like cell clusters) were observed closely associated with duct cell lining, and immunohistochemical analysis revealed that several kinds of transcription factors including PDX-1, Islet1, Nkx2.2 and Pax6 were expressed in some of duct cells and Icc cells, suggesting that Pax6 is involved in the β cell neogenesis in diabetic pancreas and the process of this type of regeneration (neogenesis) was similar to that of fetal endocrine cell development . However, Pax4 which is important for the final differentiation of β cells during development was undetectable level. Treatment with betacellulin , one of the growth factor of EGF family and is known to induce differentiation of AR42J cells into insulin-producing endocrine cells, increased the number of ICCs and ameliorate the glucose intolerance. To clarify the role of Pax4 and Pax6 in the β cell differentiation, we tried to make stable transformants of dominant negative form of these genes in AR42J cells, but this was difficult, and we could not analyze the roles of two Pax genes in β cell differentiation using in vitro experimental system.