基于Wnt和Notch信号通路探讨肠道干细胞在PI-IBS嗜铬细胞增生中的作用

Intestinal enterochromaffin cell hyperplasia plays an important role in the pathogenesis of PI-IBS, but the underlying mechanism has not been fully identified. Previous studies have shown that inflammation and stress induced enterochromaffin cell hyperplasia may have correlation with the alterations in proliferation or differentiation of intestinal stem cells, and inflammation and stress also occurred throughout the period of PI-IBS development. Therefore, we hypothesis that the alterations in proliferation or differentiation of intestinal stem cells may be involvoed in the pathogenesis of enterochromaffin cell hyperplasia of PI-IBS. In this present study, animal models of acute colitis and PI-IBS will be established; immunohistochemistry and Western-blot techniques will be used to evaluate the proliferation and differentiation of intestinal stem cells; Western-blot and Q-PCR techniques will be applied to determine the alterations in Wnt/Lrp5(6)/β-catenin and Notch signaling pathways, which are the main pathways regulating proliferation and differentiation of intestinal stem cells. Focused on the development period from acute colitis to PI-IBS, this study will investigate the proliferative and differentiative feature of intestinal stem cells, and identify their role in the pathogenesis of enterochromaffin cell hyperplasia. We predicted that the preliminary resluts from this study will give clues for pathogenisis discovery of PI-IBS, and thus provide a new target for the prevention and treatment of PI-IBS in the furture.

嗜铬细胞增生是感染后肠易激综合征（PI-IBS）发病的关键环节，但嗜铬细胞增生的机制目前尚不明确。相关研究显示，炎症或应激引起的嗜铬细胞增生可能与肠道干细胞增殖分化异常有关，而炎症和应激反应同时存在于PI-IBS的发生过程。因而，肠道干细胞增殖和/或分化异常可能与PI-IBS嗜铬细胞增生高度相关。课题组拟以急性结肠炎和PI-IBS模型大鼠为研究对象，用免疫组化及Western-blot技术研究肠道干细胞的增殖和分化；用Q-PCR及Western-blot技术检测调控干细胞增殖分化的Wnt/Lrp5（6）/β-catenin和Notch信号中相关基因及蛋白的表达；通过分析Wnt和Notch信号通路的变化探讨干细胞增殖和/或分化改变与嗜铬细胞增生的关系。通过项目的实施，有望发现肠道干细胞在嗜铬细胞增生中的作用，可为深入探讨PI-IBS发病机制提供线索，为PI-IBS防治提供新的靶点。

肠道嗜铬细胞增生是感染后肠易激综合征（Post-infectious irritable bowel syndrome, PI-IBS）发病的关键环节，但其具体发病机制目前尚不明确。课题组基于前期研究结果，推测肠道干细胞增殖和/或分化异常可能参与PI-IBS肠道嗜铬细胞的病态增生。研究应用TNBS分别诱导建立急性结肠炎、慢性结肠炎及PI-IBS大鼠模型，用免疫组织化学法考察模型大鼠肠粘膜中分泌系细胞，即杯状细胞、潘氏细胞和内分泌细胞以及嗜铬细胞的数量和分布；实时荧光定量PCR技术检测模型大鼠肠粘膜中调控分泌系细胞分化的Notch信号相关基因的表达；用免疫印迹及实时荧光定量PCR技术研究调控肠道干细胞增殖的Wnt/LRP5(6)/β-catenin信号通路相关基因和蛋白的表达。结果显示：①急性肠道炎症可上调Wnt/Lrp5(6)/β-catenin信号通路，即LRP5、LRP6、β-catenin及cyclin D1的表达均增加；而在PI-IBS期，LRP5、LRP6、β-catenin及cyclin D1的表达均接近正常水平，提示Wnt/Lrp5(6)/β-catenin信号通路可能参与炎症状态下干细胞的增殖促并促进粘膜修复。②急性肠道炎症可激活Notch信号使其下游基因Rath1、Hes1、Gfi1、Sox9、Ngn3表达上调，Ngn3表达上调致其下游基因，如Pax6、Pax4和NeuroD的表达也显著增加；而在PI-IBS期，肠道Notch信号相关基因的表达显著低于急性炎症期，其表达也趋于正常水平。③急性肠道炎症期，结肠及小肠内分泌细胞及嗜铬细胞数量无明显改变；而在肠道炎症恢复期和PI-IBS期结肠及小肠粘膜内分泌细胞数量及嗜铬细胞数量均显著增加，并伴有潘氏细胞数量的增多，而杯状细胞数量在急性炎症期、慢性炎症期及PI-IBS期均无明显变化。由此可见，肠道急性炎症时干细胞的增殖及分化能力增强，进而导致肠道干细胞向分泌系细胞的分化过程加快；随着炎症的恢复，上调的肠道干细胞增殖及Notch信号相关基因表达逐渐恢复正常，但内分泌细胞和嗜铬细胞数量仍显著增多，提示肠道干细胞分化异常可能参与肠道内分泌细胞及嗜铬细胞的病理性增生。