miR-128调控结肠免疫微环境在慢传输型便秘发病机制中的作用

The pathogenesis of colonic slow transit constipation(STC) remains unclear.Recent studies indicate that miRNAs affect the absence or loss of ICC(interstitial cells of Cajal)and SMC(Smooth muscle cell) phenotype,which may lead to gastrointestinal motility disorders.Our previous studies have shown that the expression of miR-128 is significantly decreased in colonic tissues of patients with STC compared to normal colonic tissues, and is significantly positive correlated with the expressions of c-kit and smoothelin.In addition,increased immune cells (macrophage and mast cell) are found in the in colonic tissues of patients. Basing on the results, we bring forward a hypothesis that miR-128 may play a crucial part in the pathogenesis of STC by regulating the colonic immune microenvironment, development and maintenance of ICC and SMC phenotype.In this study,we will investigate the effects of knock-down and over-expression of miR-128 on miR-128 target genes, signaling pathway proteins,inflammatory cells,inflammatory mediators,abnormalities and damage of colonic SMC and ICC from rat colon in experimental rat model of STC. Furthermore, we will demonstrate the effects of miR-128 and target genes during colonic STC development using human colon samples obtained from patients with STC.This study will provide the theoretical basis for microRNA as new therapeutic target and diagnostic biomarker in colonic STC.

MicroRNA可能参与了胃肠动力性疾病的发病机制。我们前期研究发现：(1)慢传输型便秘（STC）结肠组织miR-128表达下降，且与Cajal间质细胞(ICC)和平滑肌细胞(SMC)正相关；(2)STC结肠组织中炎症细胞增加，与miR-128表达关系密切。由此推测：miR-128可能通过调控结肠免疫微环境参与STC发病机制。本项目以动物STC模型为研究对象，观察结肠组织内miR-128及其靶基因及相应信号通路、巨噬细胞和肥大细胞形态学、炎症介质表达等的动态变化,miR-128沉默和过表达对miR-128靶基因、信号通路、炎症细胞、ICC和SMC形态及数目的影响，探讨miR-128通过调控结肠免疫微环境参与STC的发病机制，并以人体标本验证，为miR-128成为STC诊治新靶点提供理论依据。

目的 探讨miR-128调控结肠免疫微环境中在慢传输便秘（STC）疾病发病机制的作用.实验方法 ⑴Wistar大鼠慢传输型便秘模型构建成功后。行钡剂灌胃后再次评估结肠传输情况，确证造模成功后断颈法处死老鼠，取小鼠结肠标本行形态学检查（HE及标本大体观）并分段保存结肠肠管大体标本。⑵MicroRNA芯片检测：miRNA基因谱检测采用Agilent mouse miRNA（8*60K）V21.0芯片，共计检测吗啡组、吗啡+大黄组（n=5）小鼠模型1881个成熟miRNA。⑶q-PCR法检测动物模型标本miR-128表达量。⑷q-PCR法检测生理盐水正常组（n=18）、大黄组（n=12）、吗啡组（n=18）、吗啡+大黄组（n=12）中M-CSF、MAPK炎症信号通路、炎症因子、肠神经元相关指标的表达量。⑸双向凝胶电泳（2-DE）技术，计算机辅助的图像分析以及质谱鉴定方法，对5例STC患者与5例正常人结肠组织蛋白质进行分离和比较分析。.结果 ⑴结肠传输实验证实实验组出现显著结肠传输减缓（P＜0.05），吗啡+大黄组较大黄组粪便明显干结、体积减小，HE结果示该组较对照组肠管明显变薄且肌层结构紊乱。⑵MicroRNA芯片检测结果显示：共计62个miRNA呈现差异表达（差异倍数＞2，P＜0．05），与吗啡对照组相比miR-3068-3p、miR-144-3p等共计20个miRNA表达上调，miR-7223-5p、miR-5135等共计42个miRNA明显表达下调。⑶与吗啡组相比，吗啡+大黄组e组织中M-CSF明显上调（P＜0.05）、MAPK炎症信号通路表达出现下调、炎症因子IL-6表达量显著上调（P＜0.05），TNF-α表达未见统计学差异、NSE及SCF在组织中表达下调（P＜0.05）。⑷人体组织双向凝胶电泳：正常对照组平均胶蛋白斑点数为239个，STC组平均胶蛋白斑点数为215个，与正常对照组平均胶匹配点数为197个，匹配率为82％，有14个蛋白质点的表达量差异有统计学意义（差异倍数＞10，P＜0．05），其中有12个蛋白点表达发生明显上调，有2个蛋白点表达发生明显下调.结论 成功在小鼠中构建STC模型；STC模型中miR-128表达下调；在STC模型中miR-128可通过上调炎症介质M-CSF引起结肠炎症免疫微环境紊乱。