多巴胺及其受体调节大鼠结肠黏液分泌的机制及其对结肠黏膜保护作用初探

The intestinal mucus is the first and the most immportant layer that protects gut from infection. The major compoment of colonic mucus is mucin 2（MUC2），which is mainly secreted by goblet cells.It is reported that mice lacking Muc2 easely develop spontaneous colitis. Neurotransmitters such as cholinergic agents,vasoactive intestinal peptide(VIP),nitric oxide (NO),can directly stimulate colonic mucin secretion.Dopamine(DA) as an important neurotransmitter,plays an important role in the regulation of gastrointestinal (GI) motility, GI epithelial ion transport and the protection of gastroduodenal mucosa,but the effect of DA on colonic mucus secretion and its colonic mucosal protection remain unclear.Our recent studies showed that dopamine receptor 5（D5） immunoreactivity（IR） was observed on colonic goblet cells,and in the enteric nervous system,VIP and NO neurons manifest D1 and D2 IR,respectively. In additon,the DA content in colonic mucosa and submucosal preparations was significantly increased，but the protein level of MUC2 was significantly decreased in 6-OHDA-treated rats. The present study aims to test a hypothesis that DA affect colonic mucus seretion directly or indirectly by regulating the release of neurotransmitters from enteric neural plexus. By RT-PCR and western blot method, we can determine the expression of dopamine receptors in rat colon. Using MUC2 expression as a marker for goblet cells and double labeling immunohistochemistry with DA receptor antibody, we will be able to discover the cellular localization of DA receptors in rat colonic mucosa and subumucosal plexus.DA-induced neurotransmitter release will be determined with radioimmunoassay.Colonic mucosa-only or mucosa-submucosal preparations will be incubated in vitro to determine DA-induced MUC2 release that will be determined by enzyme-linked immunosorbent assay.Inflammatory bowel disease rats will be used to investigate the effects of DA and DA receptors on colonic mucosal protection through regulating the mucus secretion.Knowledge of the cellular localization of DA receptors in the colonic mucosa and Meissnar's plexus, and the mechanism underlying the regulation of the colonic musus secretion will provide more direct theoretical and laboratory basis for the role of dopamine on gut physiological regulatory mechanisms.

黏液是肠道防御微生物入侵的第一道屏障，结肠黏液的主要成分是黏蛋白2(MUC2)，由杯状细胞分泌，MUC2异常可引发炎性肠病。多巴胺（DA）具有胃指肠黏膜保护作用，但其调节结肠黏液分泌的机制不明确。我们前期研究发现，结肠黏膜杯状细胞上有DA受体5（D5）分布，肠神经丛中VIP和NO能神经元上亦表达D1和D2；我们还发现6-OHDA帕金森病大鼠的结肠内含有较高浓度的DA，但MUC2含量明显降低，黏膜通透性显著增高。我们推测DA可能直接通过黏膜DA受体或通过肠神经系统间接调节结肠黏液的合成及分泌，并参与结肠黏膜保护。本研究拟采用分子生物学及免疫组化等技术观察DA受体在结肠各层的表达及细胞定位；功能学实验观察DA及其受体对肠神经递质及结肠黏液合成与分泌的影响；并制备炎性肠病模型进一步探讨DA及其受体的参与机制及其是否可通过黏液调节发挥黏膜保护。该研究为DA参与黏液保护屏障的调节提供实验与理论依据。

胃肠黏膜上皮机械屏障主要由黏膜上皮细胞、细胞间紧密连接及黏液层等构成，上皮屏障结构及功能异常是炎性肠病及消化性溃疡发生的重要环节。本研究对单胺类递质多巴胺（DA）及五羟色胺（5-HT）对大鼠消化道黏液、黏膜通透性及水电解质转运等方面作用进行探讨。采用realtime PCR、Western blot及激光共聚焦显微镜结合免疫组织化学双标技术检测DA受体在肠道的表达及分布。利用ELISA、阿利新蓝-过碘酸雪夫氏染色及体外孵育结肠组织检测肠黏膜cAMP及黏液MUC2的含量变化。利用荧光标记的右旋糖酐（FITC-dx4）结合短路电流等技术深入探讨DA、5-HT调控大鼠肠黏膜屏障通透性及水电解质转运机制。应用人D5受体转基因小鼠深入探讨D5受体对肠道黏液、通透性等方面的影响。水浸束缚急性应激大鼠模型用以研究应激情况下5-HT对结肠分泌的影响。结果显示DA受体D1、D2及D5在十二指肠和结肠黏膜层均有表达；以MUC2为杯状细胞标志，可发现D5受体在大鼠结肠杯状细胞上有大量分布；体外孵育功能结果显示DA可促进大鼠结肠黏多糖的分泌及MUC2和cAMP的合成，而D1类受体拮抗剂可阻断该作用。DA可刺激大鼠十二指肠黏膜短路电流及电阻降低，而通透性增加，D1类受体拮抗剂可阻断该作用。人D5受体下调转基因小鼠十二指肠黏膜基础短路电流、通透性及DA诱导的cAMP降低；人D5受体上调的转基因小鼠黏膜基础短路电流、通透性及DA刺激的cAMP含量增高，电阻和紧密连接蛋白（ZO1和Occludin）表达显著降低。应激可引起胃肠黏膜屏障的结构及功能异常，本研究同时发现急性应激大鼠模型结肠黏膜的基础电流和基础cAMP含量均较正常大鼠明显升高；对5-HT诱导的促分泌作用及cAMP水平也显著增高，该作用可被5-HT4受体拮抗剂完全阻断，而5-HT4受体的表达量并无改变，5-HT在应激大鼠黏膜引起的增高的cAMP水平与正常结肠黏膜相比无明显差异。结论：DA可通过D5受体及cAMP的介导促进结肠黏液分泌及通过下调紧密连接蛋白提高十二指肠的黏膜通刺激透性；应激增加的大鼠结肠黏膜cAMP含量可增强5-HT的促黏膜分泌作用。以上研究为单胺类递质参与消化道黏膜上皮屏障的调节提供新的思路和实验室依据。