关键分子表达调控异常及表观遗传学改变在糖尿病小鼠小肠上皮干细胞增殖/分化障碍中的作用

Diabetic enteropathy was previous regarded as the disease which had a connection with the abnormal motor function of intestine and studies on its mechanism were fouced on the functional alterations of interstitial cells, such as cajal cells. However, it could not explain the new discovery that intestinal barrier was impaired and epithelium was dysplastic in diabetic enteropathy. Especially, the mechanisms and impact of hyperglycemia on intestinal epithelial stem cells (IESC) are not clear. In our project, we will take our pre-experiment results, which have indicated abnormal proliferation and differentiation of IESCs and aberrant expressive trend of Msi1 and Hes1 in diabetic mice, as the starting point. Meanwhile, we will establish the diabetic enteropathy mouse model and primary culture system for intestinal epithelial cells and IESCs in vitro. RNA in situ hybridization, quantitative PCR, Western blots, gene silencing, promoter elements analysis, promoter methylation, etc. will be applied in this study. In the view of alteration in related signaling pathways and epigenetics, we will systematically investigate the mechanism of abnormal proliferation and differentiation, and explore the new targets and methods to recover the proliferation and differentiation status of IESCs in the diabetic mouse. This project will be destined to have great theoretical significance and application value, because we are aimed both at providing new methods to improve the intestinal epithelial function of diabetic enteropathy.

糖尿病肠病既往被认为与肠道运动功能异常有关，其机制研究也集中在探讨Cajal细胞等间质细胞的功能改变。但新近发现糖尿病的肠上皮增生异常，肠屏障功能受损，这些均难以用上述机制所解释，尤其高血糖对肠上皮干细胞（IESC）的具体影响及机制尚不明确。本课题以预实验中发现的糖尿病小鼠IESC增殖/分化异常及Msi1、Hes1表达趋势异常为切入点，建立小鼠糖尿病肠病模型和原代肠上皮细胞及IESC的体外分离、培养体系，利用RNA原位杂交、定量PCR、Western blots、基因沉默、启动子元件分析、启动子甲基化检测等方法，从相关信号通路异常及表观遗传学改变这一新角度，系统全面的探讨糖尿病小鼠肠上皮增殖、分化及功能异常的机制，探寻恢复IESC增殖/分化状态的新靶点及有效方法，在完善糖尿病肠病发病机制的同时，也为改善糖尿病肠病患者肠道上皮增殖/分化状态及肠功能提供新的手段，具有重大的理论意义及应用价值。

目前，糖尿病肠上皮增殖/分化改变的机制并没有阐明，而作为肠上皮各类型细胞来源的肠上皮干细胞在糖尿病状态下的增殖/分化改变更是少有报道。本课题系统阐明了：（1）糖尿病状态下，肠上皮增殖/分化的改变，并且发现糖尿病小鼠肠屏障功能异常和肠上皮分化异常是通过抑制Notch/Hes1信号通路中的Notch/NICD转导的；（2）糖尿病状态下，MiRNA-30e对肠上皮增殖/分化的作用，并且发现在糖尿病小鼠中MiRNA-30e通过下调Dll4来调控小肠上皮细胞的异常分化；（3）糖尿病状态下，转录因子Sox9对肠上皮增殖/分化的作用，并且发现了在糖尿病小鼠中，隐窝区域细胞的基因启动子甲基化整体是下降的，Sox9通过Wnt通路调控肠上皮干细胞的异常增殖。以上的研究对于阐明糖尿病状态下肠上皮细胞的增殖/分化改变起到重要的作用，为进一步探讨改善糖尿病肠道症状的机制和方法提供了实验理论基础。