基于c-RET介导NF-κB的自噬激活机制在先天性巨结肠发病中作用的研究

Hirschsprung’s disease is a common congenital gastrointestinal malformation, characterized by the lack of ganglion cells driven from enteric neural crest stem cells (ENCSCs). The most frequent cause gene of Hirschsprung’s disease is c-RET. c-RET can stimulate the phosphorylation of NF-κB and NF-κB is involved in autophagy have been reported. But it has not been determined whether c-RET initiates autophagy via NF-κB to promote the differentiation of ENCSCs. In our previous results, we found calpain1, Atg5 both expressed in Hirschsprung’s disease, and the expression level of Calpain1 was inversely correlated with Atg5. In vitro study also showed that the cleavage of Atg5 could be decreased by downregulation of Calpain1 and then promoted the differentiation of ENCSCs/ENPCs. This study is to further investigation on the mechanism of autophagy triggered by c-RET via NF-κB mediated Calpain1 and its regulation in the differentiation of ENCSCs/ENPCs. Whereafter, we will set up c-RET-KIY1062F/Y1062F and c-Ret-KIY1062F/+;RET-Tg mice to prove it in vivo. Transplantation of modified ENCSCs/ENPCs or stimulation the differentiation in situ will be done to renovate the lack of ganglion cells. Taken together, this project will further elucidate the role of autophagy in Hirschsprung’s disease triggered by c-RET and also provide a potential treatment strategy.

先天性巨结肠是病变肠壁肠神经嵴干细胞来源的肠神经节细胞缺如所致的常见先天消化道畸形，c-RET为最重要的致病基因，可能通过介导磷酸化激活NF-κB，启动细胞自噬从而促进肠神经嵴干细胞的分化。我们前期研究发现，NF-κB下游调控基因Calpain1、Atg5均表达于先天性巨结肠切除肠管的肌间神经丛，并且Calpain1的表达与Atg5呈负相关，体外实验证实下调Calpain1可以减少Atg5的剪切，并促进了肠神经嵴干细胞/前体细胞的分化。本课题是前期工作的深入，将阐明c-RET如何通过介导NF-κB调控Calpain1并激活自噬，从而促进肠神经嵴干细胞分化，并构建c-RET-KIY1062F/Y1062F和c-Ret-KIY1062F/+;RET-Tg模型鼠验证，进行诱导及原位移植，从细胞自噬角度阐明c-RET缺陷导致先天性巨结肠发病的分子基础并探讨潜在的治疗策略。

先天性巨结肠是病变肠壁肠神经嵴干细胞来源的肠神经节细胞缺如所致的常见先天消化道畸形，c-RET为最重要的致病基因，本研究发现Yap1可以影响c-RET的表达，通过介导磷酸化激活NF-κB，启动细胞自噬从而促进肠神经嵴干细胞的分化。前期的研究我们发现NF-κB下游调控基因Calpain1、Atg5均表达于先天性巨结肠切除肠管的肌间神经丛，并且Calpain1的表达与Atg5呈负相关，体外实验证实下调Calpain1可以减少Atg5的剪切，并促进了肠神经嵴干细胞/前体细胞的分化，而本课题进一步的研究发现c-RET介导的细胞自噬途径，不是通过Calpain1调控，而是通过NF-κB直接作用于细胞自噬基因，从而影响肠神经嵴干细胞的分化，为探讨先天性巨结肠的发病提供了分子基础。在此基础上，课题组以肠神经系统模型鼠Wnt1-tdTomato转基因鼠为模型，构建了小鼠胚胎活体成像系统，并在嵌合体中研究了人鼠神经嵴细胞的嵌合差异，为本课题的进一步深入提供了基础。