FoxO1在胰岛细胞定向分化和功能重建中的作用及机制研究

Many studies have shown that FoxO1 participates in islet development and stem cell differentiation. Recent publications reported that mouse gut endocrine progenitor cells could be differentiated into the glucose-responsive, insulin-producing cells by Foxo1 ablation. Moreover, FoxO1 inhibition could yield functional insulin-producing cells in the human gut organoids derived from human induced pluripotent stem cells. Our preliminary data showed that an active small-molecule inhibitor of FoxO1 promoted human embryonic stem cells to differentiate into islet-like cells. Based on these observations, we hypothesize that FoxO1 inhibits islet cell differentiation from stem cells (in vitro) and islet function reconstruction (in vivo) by regulating relative signaling pathways and/or pancreas-specific transcription factors during the differentiation. In this research project, we intend to induce FoxO1 inhibition or over-expression in the different stages of islet cell differentiation from either human embryonic stem cells or human pancreatic progenitors, to detect the effects of FoxO1 on islet cell differentiation, and to determine the regulatory roles of FoxO1 on the relevant signaling pathways and potential target genes during the differentiation. Furthermore, we will also investigate the efficacy of FoxO1 inhibition as a therapeutic target in treating the mouse models of type 2 diabetes, and determine the roles of FoxO1 inhibition in the trans-differentiation of gut endocrine progenitors and the islet function reconstruction in these animals. Our project is helpful not only for clarifying the roles and mechanism of FoxO1 in islet cell differentiation, but also for promoting the islet cell regeneration in vivo as a therapeutic strategy in treating type 2 diabetes.

业已证实，FoxO1参与胰岛胚胎发育和干细胞定向分化。近期另有研究显示，抑制FoxO1可促进肠道内分泌前体细胞或干细胞来源的肠道样器官向胰岛细胞转化。我们的预实验结果显示，FoxO1小分子抑制剂可促进人胚胎干细胞向胰岛细胞分化。由此我们假设：FoxO1通过调控分化相关信号通路和（或）胰腺特异性转录因子，抑制干细胞向胰岛细胞分化，抑制体内胰岛功能重建。在前期工作基础上，本项目拟在人胚胎干细胞和人胰腺前体细胞向胰岛细胞分化的不同阶段抑制或过表达FoxO1，明确其对胰岛细胞定向分化的影响，并且确定FoxO1对细胞分化相关信号通路和靶基因的调控作用。此外，本项目还将观察抑制FoxO1在2型糖尿病小鼠中的治疗作用，探讨其在肠道内分泌前体细胞转分化和胰岛功能重建中的作用。本项目不仅有助于阐明FoxO1在胰岛细胞定向分化中的调控作用及其机制，而且为研发促进体内胰岛再生的治疗策略提供实验依据和理论基础。

已有报道显示，FoxO1参与胰岛胚胎发育和干细胞定向分化，抑制FoxO1可促进肠道内分泌前体细胞或干细胞来源的肠道样器官向胰岛细胞转化。本课题进一步明确了FoxO1对人胚胎干细胞细胞和人胰腺前体细胞向胰岛细胞定向分化的影响，阐明了其具体作用机制，并探讨了抑制FoxO1 对2 型糖尿病小鼠的治疗作用及其相关机制。结果显示：在人类胚胎干（hES）细胞和单克隆人胰腺前体细胞系向胰岛素产生细胞分化过程中，在分化的各阶段中均可检测到FoxO1在胞浆和胞核的表达。FoxO1敲减可上调胰腺前体细胞和成熟胰岛细胞标志物，并促进胰岛素分泌，而过表达FoxO1则发挥相反的效应，提示抑制FoxO1可促进hES细胞向胰岛细胞分化和成熟。添加FoxO1抑制剂AS1842856可促进FoxO1磷酸化，上调胞浆FoxO1的表达，胞核FoxO1未见明显变化，提示小分子抑制剂可能通过调控FoxO1的定位和转录活性参与胰岛细胞的分化。我们在hES细胞分化不同阶段开展FoxO1的染色质免疫共沉淀（ChIP）实验，并进行高通量测序筛选及验证，发现FoxO1能够直接调控细胞周期相关蛋白cyclin D2。在hES细胞向β细胞定向分化过程中添加cyclin D2抑制剂能够抑制β细胞分化，提示cyclin D2可能参与FoxO1介导的hES细胞向胰岛细胞的定向分化。与此同时，我们将hES细胞分化来的胰腺前体细胞移植到NOD/SCID小鼠肾包囊下，发现抑制FoxO1同样能够促进β细胞在体内的分化成熟。此外，我们成功构建了β细胞特异性敲除FoxO1小鼠（Ins2-FoxO1-/-）和β细胞谱系示踪小鼠，明确在糖尿病发生发展过程中，FoxO1与胰岛β细胞功能变化之间的关系。本研究在国内外首次明确了FoxO1在胰岛细胞定向分化和功能重建中的作用及其潜在的作用机制，为研发促进体内胰岛再生的治疗策略提供实验依据和理论基础。本项目已发表论著26篇，其中SCI论文16篇，中文核心10篇。研究成果在全国性大型学术会议上做大会报告、特邀报告或口头发言10余次。培养研究生9名，其中博士6名，硕士3名，出站博士后1名。