中文摘要：

胰岛功能受损的分子机制研究是揭示2型糖尿病（T2DM）发病机制的核心问题.FOXO1是胰岛素信号下游的重要靶转录因子,参与胰岛的发育,但在分化成熟的胰岛β细胞中的功能尚未阐明.本研究采用免疫组化方法结合激光共聚焦技术观察FOXO1在胰岛的表达及细胞定位;通过基因介导的转移技术和siRNA干预技术,在培养的大鼠胰腺癌β细胞系（INS-1E）中特异高表达组成性活性的FOXO1（FOXO1-AAA）或抑制其表达水平,观察FOXO1表达水平的改变对β细胞增殖、凋亡的影响.免疫组化结果显示,FOXO1在正常胰腺组织中仅特异地表达在胰岛内.采用胰岛素与FOXO1的免疫荧光双标结合共聚焦观察进一步揭示,FOXO1主要表达在胰岛的β细胞中.Western印迹显示,腺病毒介导的基因转移技术在体外培养的INS-1E细胞中过表达FOXO1-AAA或其特异的siRNA均能有效地上调或抑制其表达水平.^3 H-TdR掺入实验结果显示,降低FOXO1的表达显著促进细胞增殖;反之,高表达FOXO1显著抑制细胞增殖.与之相应,MTT检测结果显示,降低FOXO1的表达对细胞存活有显著促进作用,高表达FOXO1对细胞存活有显著抑制作用.进一步采用流式细胞仪检测细胞凋亡,结果显示降低FOXO1的表达使β细胞凋亡率降低,反之高表达FOXO1使β细胞凋亡率增加.研究结果证实,胰岛β细胞中的FOXO1参与β细胞的存活、增殖、凋亡的调节.病理性高表达FOXO1可能通过阻止β细胞增殖、促进β细胞凋亡从而减少β细胞的数量,在T2DM发生中可能起重要作用.

英文摘要：

The dysfunction of pancreatic islet β cell is a major focus in the pathogenesis of type 2 diabetes （T2DM）. FOXO1, one of the forkhead transcription factors subfamily members, is an important target downstream of insulin signaling pathway and plays crucial roles in the development of pancreatic islet. However, the exact function of FOXO1 and its regulation in pancreatic β cells remains to be further characterized. In this study, the expression and cellular localization of FOXO1 were detected by immunohistochemistry and laser confocal microscopy. Adenoviral vectors expressing a constitutively active form of FOXOI （FOXO1-AAA） and its small interfering RNA （siRNA） were prepared. The effects of FOXO1 on cell proliferation and apoptosis were investigated with rat insulinoma cell line （INS-1E） by introducing either active FoxOl or its siRNA. The results showed that FOXO1 was detected in pancreatic islet by immunohistochemistry and co-localized with insulin in 13 cells by confocal microscopy. Western blot demonstrated that the FOXO1 expression was remarkably increased by Ad-FOXOI-AAA infection and suppressed by Ad-siRNA-FOXO1 in INS-1E cells. 3H-TdR incorporation experiment showed that the INS-1E cell growth was significantly promoted by decreasing FOXO1 expression, whereas inhibited with increased FOXO1 expression. The survival of INS-IE cells detected by MTr showed similar effects. Furthermore, fluorescent activated cell sorting （FACS） analyses showed that increased apoptosis was observed at higher levels of FOXO1 expression but decreased lower FOXOI levels. These data demonstrated that FOXO1 might participate in the regulation of survival, proliferation and apoptosis of pancreatic β cells.