TSC2/mTORC1轴介导呕吐毒素损伤仔猪小肠干细胞扩增的机制及营养调控

肠道干细胞扩增是驱动小肠上皮隐窝绒毛轴更新的动力。饲料源呕吐毒素（DON）污染，降低肠道干细胞活性，抑制肠上皮发育，但其确切的机制尚不清楚。本项目在前期工作基础上，围绕TSC2/mTORC1整合其上游Frizzled7-GSK3β、IR-PI3K-Akt、和EGFR-MEK-ERK 信号，以DON处理仔猪和干细胞3D培养模型，流式细胞术探究干细胞命运决定；iTRAQ筛查响应的信号通路；免疫组化和蛋白质印迹，确认各信号通路之间的互作关系；过表达/RNAi或激动/抑制剂处理，明确候选靶标的作用机理；分子对接和修饰组分析，结合点突变验证，探明DON作用于膜受体的靶点；解析TSC2作为整合器，调控mTORC1介导DON损害干细胞扩增的机制，以全新视角丰富黏膜损伤理论体系。选用谷氨酸/酶解小麦蛋白质进行干预，探讨其是否可重新激活mTORC1，逆转DON诱导的肠道损伤，为饲料中DON污染防控提供依据。

The crypt-villus axis renewal is driven by the intestinal stem cell (ISC) expansion in the small intestine. Deoxynivalenol (DON) contamination of feed reduces the ISC activity, thus inhibiting the intestinal epithelial development; however, the exact mechanisms involved remains unclear. Based on our previous studies, the proposal focuses on the TSC2/mTORC1 axis integrated its upstream Frizzled7-GSK3β, IR-PI3K-Akt and EGFR-MEK-ERK signals in the models of DON-treated piglets in vivo and porcine ISC three-dimensional (3D) culture system ex vivo. Firstly, the injury of small intestinal epithelium will be observed by HE staining and electron microscopy, and the ISC fate decision will be investigated by flow cytometry. Subsequently, iTRAQ is used to screen the response signaling and their interaction among candidate targets is analyzed via immunohistochemistry and Wes. Moreover, the gene overexpression/RNAi or agonist/inhibitor treatment of ISC will be constructed to identify the function of candidate targets. Meanwhile, the binding sites of DON acting on membrane receptor (Frizzled7, IR and EGFR) are further confirmed by molecular docking and modification-specific proteomics, as well as point mutation for verification. The precise mechanism of TSC2, as an integrator, regulating mTORC1-mediated ISC injury induced by DON will be clarified. Finally, glutamate/hydrolyzed wheat gluten intervened to explore whether they can reactivate mTORC1 to reverse DON-induced intestinal damage. The results will enrich the theoretical system of mucosal injury from a new perspective and can be used as a basis for the prevention and control of DON pollution in the feed.