小分子RNA调控机制在糖尿病小鼠小肠上皮干细胞增殖/分化异常中的作用

Diabetic enteropathy was previously regarded as a disease which had a connection with the abnormal motorial function of intestine, and the studies on its mechanism were focused 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 this study, we will take our pre-experiment results, which indicated abnormal proliferation and differentiation of IESCs, aberrant Notch/Hes1 signal pathway in diabetic mice, and up-regulation of miR-23b amd miR-30e as the starting point. Meanwhile, we plan to establish a mouse diabetic enteropathy model and primary culture system for intestinal epithelial cells and IESCs in vitro. RNA in situ hybridization, quantitative PCR, and Western blots will be applied in the signal pathway. And the microRNA gene chip detection, PAGE/Northern blotting, and target gene analysis will be performed in microRNA mechanism research. In the view of alteration in related signaling pathways and microRNAs, we will systematically investigate the mechanism of abnormal proliferation and differentiation of IESCs in diabetic mice, and explore the new targets and methods to recover the proliferation and differentiation status of IESCs. This research project will provide theoretical and applied value for diatetes study and a new method to improve the intestinal epithelial function in diabetic enteropathy.

糖尿病肠病（DE）既往被认为与肠道运动功能异常有关。然而，近年来的研究发现DE的肠上皮呈现增生异常和肠屏障功能受损，这些均难以用上述运动异常机制所解释，尤其高血糖对肠上皮干细胞（IESC）的影响及其机制尚不明确。本课题以预实验中发现的DE小鼠IESC增殖/分化异常、Notch通路改变和miR-23b、miR-30e表达上调为切入点，通过建立小鼠DE模型和原代IESC组分的体外分离、培养，系统研究DE小鼠IESC的增殖/分化信号通路改变的情况，并采用miRNAs基因芯片筛查、Northern blot验证及靶基因研究的策略，从miRNAs调控机制这一新角度系统全面的探讨DE小鼠IESC增殖、分化改变及肠上皮功能异常的机制，尝试以关键miRNAs为调控新靶点，恢复DE小鼠IESC增殖/分化状态。在完善DE发病机制的同时，也为改善DE患者肠道上皮功能提供新的手段，具有重大的理论意义及应用价值。

糖尿病（DM）是表现为胰岛素缺乏/抵抗和高血糖的一组代谢性疾病。我们之前已经报道在1型DM小鼠的小肠上皮细胞间存在紧密连接受损，以及紧密连接蛋白Ocln和ZO-1的表达减低。同时，Notch/Hes1信号通路受到抑制可导致DM状态下的小肠上皮细胞出现异常分化。然而，这些蛋白的表达异常及通路异常至今仍未被阐明。在本次研究中，我们系统的从miRNA调控的角度研究了miRNA在DM小鼠小肠上皮细胞的紧密连接受损中的作用及机制，以及miRNA异常表达对Notch/Hes1信号通路和肠上皮分化状态的影响。采用集成比较miRNA微阵列技术，我们发现了miR-429在1型DM小鼠的小肠上皮细胞中的表达上调，证实了miR-429对Ocln 3′端非翻译区的直接靶向作用，但是并未发现其对ZO-1表达的调控作用。进一步，我们还验证了miR-429在离体IEC-6细胞中可下调Ocln的表达，并发现外源性agomiRNA-429在正常小鼠体内也可下调Ocln的表达，同时诱导小肠屏障功能异常的发生。此外，通过生物信息学分析，我们发现了miRNA-30e的表达在DM小鼠中也呈现异常增高，且可能对Notch信号通路发生调节作用。结果显示，DM小鼠小肠上皮细胞的异常分化与Dll4/NICD/Hes1信号通路受抑制相关。MiRNA-30e的靶基因位于原代小肠上皮细胞以及IEC-6细胞Dll4基因的3′端非翻译区，并可下调Dll4基因的表达。外源性miRNA-30e可减弱Dll4/NICD/Hes1信号通路的活性，诱导正常小鼠小肠上皮细胞呈现与DM小鼠相似的异常分化特征。反之，在DM小鼠体内注射miRNA-30e抑制物可上调肠上皮细胞中Dll4/NICD/Hes1信号通路的活性，使小肠上皮细胞的分化正常化。综上，miR-429的增加可通过靶向作用于Ocln 3′端非翻译区，下调Ocln的表达，进而损伤DM小鼠的小肠屏障功能。而增高的miRNA-30e可通过靶向作用于Dll4基因的3′端非翻译区，降低Dll4/NICD/Hes1信号通路活性，从而对DM小鼠小肠上皮的异常分化起促进作用。