DNMT3A调控Notch信号通路对人肠神经嵴干细胞定向分化的作用及机制研究

Hirschsprung's disease is the common disorder of digestive tract in children. At present, the major focus on how to accurately regulate GNCSC directional differentiation in the receptors whethercompletely reconstruct functional neural network by stem cell of replacement therapy. Notch signaling pathway is the key pathways to decide the fate of neural stem cells. DNA methyltransferases 3A is involved in regulating the expression of specific genes. The bioinformatics analysis and the research indicated that DNMT3 could regulate Notch gene expression. This research through the use of DNA methylation chip, to detect the DNA methylation of the GNCSC. Use of sh-DNMT3A, transplantation GNCSC to HSCR model to observe the level of methylation，cells types and the function of colon, further analysis of DNMT3A and GNCSC related gene expression level in Notch signaling pathway，the relationship between the level of DNA methylation and cellular differentiation. Its purpose is to verify the DNMT3A in Notch signaling pathways regulating GNCSC cell directional differentiation in function and mechanism. Illustrate the relationship between DNA methylation and gene expression as well as the control mechanism during the differentiation of hGNCSC, to provide new research approach and evidence for repairing ENS system by stem cells therapy in clinic.

先天性巨结肠是小儿常见消化道畸形，精确调控肠神经嵴干细胞(Gut neural crest stem cells, GNCSC) 定向分化能力是细胞移植替代治疗重建功能性肠神经网络的关键。Notch 信号通路是GNCSC“细胞命运决定”的主要环节，DNA甲基化转移酶之一DNMT3A在干细胞定向分化扮演重要角色。我们应用生物信息学分析及体外实验提示DNMT3A能够调控Notch通路基因表达。本研究拟通过DNA甲基化芯片检测不同部位GNCSC的DNA甲基化差异，转染sh-DNMT3A并移植至动物模型，体内外观察甲基化水平、细胞分化及肠功能，进一步分析DNMT3A、Notch通路相关基因表达与GNCSC关系。旨在明确DNMT3A通过Notch信号通路调控GNCSC定向分化作用，从而在理论上阐明DNA甲基化和基因表达对GNCSC的调控机制，为临床自体移植修复肠神经系统提供新的研究思路和实验依据。

先天性巨结肠是小儿常见消化道畸形，精确调控肠神经嵴干细胞(Gut neural crest stem cells, GNCSC) 定向分化能力是细胞移植替代治疗重建功能性肠神经网络的关键。Notch 信号通路是GNCSC“细胞命运决定”的主要环节，而长链非编码RNA (Long noncoding RNAs, lncRNAs)已被广泛应用于多种人类疾病。然而是否与肠神经嵴干细胞的表型(ENCSCs)相关仍不清楚。因此，在实验中我们设计了致病性AFAP1-AS1 HSCR，利用微阵列分析和生物信息学工具在不同HSCR病人进行筛选不同lncRNAs和小分子核糖核酸(microrna)，利用小干扰RNA转染观察ENCSCs能力。通过CCK-8、EdU、transwell化验和流式细胞术进行细胞体外增殖、迁徙等检测。最后,rescue实验初步分析出AFAP1-AS1和mir -195以及mir - 195和E2F转录因子3 (E2F3)协同能力，得出HSCR患者的AFAP1-AS1表达降低。同时敲除AFAP1-AS1能够移植ENCSCs的细胞体外迁移和增殖能力，并且促进ENCSCs细胞凋亡。当miR-195表达上调时，E2F3转录因子消失。而AFAP1-AS1能够抑制E2F3绑定mir -195的能力。总之，AFAP1-AS1沉默作为内源性RNA与mir - 195作用，能够改变E2F3表达从而抑制ENCSC的活动能力并导致HSCR。明确了IncRNA在Notch信号通路调控GNCSC增殖能力作用，从而在理论上阐述了在IncRNA及microrna水平对GNCSC的调控机制，为临床自体移植修复肠神经系统提供新的研究思路和实验依据。