转录因子NGN3/PDX1/MAFA诱导人胃肠类器官重编程为胰岛素分泌细胞的方法和机制初探

胃肠干细胞具有自我更新和多分化能性，小鼠中应用beta细胞重编码因子NGN3, PDX1, MAFA(NPM)可促进胃肠干细胞转分化为胰岛素分泌细胞，可望成为beta细胞新的再生来源。但人胃肠干细胞能否重编程为胰岛beta细胞，目前尚无报道。本课题组在前期研究中首次提出“胃肠干细胞-胃肠内分泌细胞-产胰岛素细胞”的分步诱导方案，在人胃肠干细胞中先诱导NGN3促进内分泌细胞分化, 继而PDX1/MAFA 过表达能够明显提升胰岛素的表达。本课题拟在此基础上，进一步探索三种转录因子以不同启动方式对细胞转分化的影响，创新性应用胃肠类器官培养技术和营养节律性培养驯化生物钟促进其功能成熟，优化实验方案。更进一步应用单细胞转录组测序技术对处于分化不同时间点的细胞进行检测，解析细胞转分化的过程，探索NPM介导的转录调控网络在触发干细胞分化的关键机制，为实现糖尿病的自体细胞移植治疗提供新的突破。

Gastrointestinal stem cells are capable of self-renewal and multi-differentiation. The application of beta cell reprogramming factors NGN3, PDX1 and MAFA (NPM) in mice can promote the transformation of gastrointestinal stem cells into insulin-secreting cells, which is expected to be the new regeneration source of beta cells. Whether human gastrointestinal stem cells can be reprogrammed into islet beta cells has not been reported. In the previous study, our group proposed for the first time a step-by-step induction scheme of "gastrointestinal stem cells - gastrointestinal endocrine cells - insulin-producing cells". In human gastrointestinal stem cells, NGN3 was first induced to promote the differentiation of endocrine cells, and then the overexpression of PDX1/MAFA could significantly improve the expression of insulin. On this basis, this project intends to further explore the influence of three transcription factors on cell transdifferentiation in different modes, and innovate the application of gastrointestinal organ culture technology and nutritional rhythm culture to entrain circadian clock to promote its functional maturity, so as to optimize the experimental scheme. Furthermore, single cell transcriptome sequencing technology was further applied to detect cells at different time points of differentiation, to analyze the process of cell transdifferentiation, to explore the key mechanism of NPM-mediated transcriptional regulation network in triggering stem cell differentiation, and to provide a new breakthrough for the realization of autologous cell transplantation cell therapy for diabetes.