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，涉及多种调节生长和分化因子表达的转录因子。其中，已知Pax家族在胰岛素分泌β细胞的最终分化中起重要作用。另一方面，在β细胞衰竭的糖尿病胰腺中发生的新生机制知之甚少。因此，我们研究了选择性四氧嘧啶灌注诱导葡萄糖不耐受小鼠胰腺β细胞新生的过程，特别参考了Pax家族等转录因子的表达。我们还试图通过引入AR42J细胞的显性阴性形式来阐明Pax 6和4的作用。胰岛样细胞群（ICCs）与胰管细胞内层密切相关，免疫组化分析显示，部分胰管细胞和胰管细胞中表达PDX-1、胰岛样细胞1、Nkx2.2、Pax6等转录因子，提示Pax6参与了糖尿病胰腺β细胞的新生，这种再生（新生）过程与胎儿内分泌细胞的发育过程相似。然而，在发育过程中对β细胞最终分化至关重要的Pax4未检测到。β细胞素是EGF家族的生长因子之一，已知可诱导AR42J细胞分化为产生胰岛素的内分泌细胞，增加ICCs的数量并改善葡萄糖耐受不良。为了明确Pax4和Pax6在β细胞分化中的作用，我们试图在AR42J细胞中构建这些基因显性阴性形式的稳定转化子，但这很困难，并且我们无法使用体外实验系统分析两个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.