5-HT调控仔猪肠上皮细胞更新及紧密连接蛋白Occludin表达的"5-HTR—VIP"能神经通路

With the intensive livestock development and stresses factors increasing, the study on nerve regulation of intestinal function abnormal becomes to the "hot spot" in the field of life science. 95% 5-HT locates in the gastrointestinal tract under physiological status, but it increases and signal transmits abnormally under stress, which will cause diarrhea. Previous study find that 5-HT、VIP nerve are rich in intestinal mucous membrane, and both are positive correlation. Intestinal epithelium is an important unit of intestinal mucous barrier. However, the regulation of 5-HT on intestinal epithelium structure and function is still unclear. Therefore, we propose the assume that the "receptor- nerve" pathway of 5-HT on the IEC renovation and cell junction. The project explore to ultrastructure and cell activation of IEC, the expression of tight junction protein (occludin),5-HT receptors and the proliferation and differentiation of IEC in intestinal crypt by use of the neurotransmitter adding, nerve receptors blocking, the incorporation of in vivo and in vitro and the diarrhea model of piglet. It is expected that the regular pattern development and molecule foundation of the intestinal mucosal barrier reconstruction is resolved roundly, and next the mechanism of stress diarrhea is clearer in the future.

随着集约化养殖和环境应激因素增加，应激性胃肠紊乱的神经调控成为生命领域研究的"热点"。机体95%的5-HT存在于胃肠道，应激引起5-HT增加，信号传导异常，导致腹泻。前期研究发现肠黏膜中富含5-HT、VIP能神经纤维，二者呈正相关。肠上皮细胞作为肠黏膜屏障的重要组成单位，5-HT调控肠上皮细胞结构、功能的研究很少。因此，本项目提出5-HT调控肠黏膜上皮细胞更新和细胞连接构建的受体-神经作用通路的设想。本课题利用添加递质、受体阻断、体内与体外实验、仔猪感染腹泻等实验手段，检测肠黏膜上皮超微结构与细胞生物活性、T-J蛋白occludin的表达、5-HT受体以及肠隐窝上皮细胞增殖分化，阐明5-HT调控肠黏膜上皮细胞更新和紧密连接蛋白表达的"5-HTR-VIP"能神经通路。以期全面解析神经调控肠黏膜屏障建设的动态变化规律和分子基础，揭示应激性腹泻的发生机制，为应激性胃肠功能紊乱的诊治提供新的突破点

本项目建立应激腹泻模型及5-HT增加和降低模型，结合体内和体外实验，利用组织学、组织化学、ELISA、流式细胞术及Western Blot等实验技术，检测应激腹泻和5-HT、VIP对肠黏膜机械屏障和免疫屏障的影响，探讨神经递质在断奶仔鼠应激腹泻发生中的作用和调控机制。结果如下：1.仔鼠断奶应激伴随肠道和血浆5-HT含量升高，而肠道5-HT含量升高也会导致或者加剧断奶仔鼠应激腹泻程度；同时，VIP在肠道中的表达量与腹泻和5-HT表达量呈现高度相关。2.应激腹泻组体重、小肠绒毛高度显著下降，隐窝深度不同程度增加，V/C平均比值显著降低，肠腺PCNA表达降低；空肠、回肠和结肠紧密连接蛋白表达显著降低；5-HT降低能改善应激腹泻引起的肠道组织学病变，而5-HT含量增加能引起应激腹泻相似的肠黏膜损伤。3．应激腹泻组，肠段淋巴细胞增殖活性及细胞因子在肠道的分布形式发生改变；抗氧化能力下降，氧化产物增加，打破了肠道黏膜免疫平衡状态；而5-HT降低能改善应激腹泻引起的肠道免疫水平紊乱和氧化应激水平。4.低浓度5-HT能促进IEC-6细胞系增殖，抑制细胞凋亡；高浓度5-HT可能通过刺激并结合细胞表面的5-HT3R，激活细胞内ERK途径，最终激活凋亡效应器的终端，引起cleaved caspase-3表达量增加，从而促进IEC-6细胞系的凋亡。5.在5-HT介导断奶应激腹泻的过程中，VIP能神经元通过其表达的5-HT3R发挥重要的作用；VIP 主要通过VPAC1呈剂量依赖式的促进IEC-6细胞增殖，抑制IEC-6细胞的凋亡。综上所述，本项目旨在探讨神经递质在应激腹泻中对肠黏膜机械屏障、免疫屏障及抗氧化功能的影响，揭示5-HT和VIP对胃肠道功能的调节作用，进一步阐明肠黏膜屏障建设的动态变化规律及分子基础，加深对应激性腹泻病程发生机制的认识，同时也为防治“断奶仔猪应激腹泻”提供一定的理论依据。