上皮转录因子p63与增强子调控鼻黏膜炎症上皮干细胞异常增殖和分化的机制研究

鼻黏膜上皮修复和重塑异常导致黏膜炎症发生发展。申请人已明确上皮转录因子（p63）在炎症上皮修复下降和异常重塑起关键作用（Thorax; JACI）；通过建立鼻上皮干细胞体系，发现鼻息肉上皮干细胞（p63+）体外增殖能力下降和分化异常（Sci Report;JACI）。但p63调控上皮干细胞增殖和分化的机制不明。预实验表明鼻息肉上皮干细胞增殖过程中，p63和增强子（提高转录频率的染色质元件）的基因调控活跃度低于正常干细胞，且与细胞周期和增殖基因表达相关。我们假设：炎症鼻上皮干细胞转录因子p63和增强子协同调控下游基因的机制被改变，导致细胞异常增殖和分化。本项目通过培养正常和炎症鼻黏膜上皮干细胞，采用ChIP-seq技术分析p63在染色质与增强子结合位点，利用RNA-seq检测基因转录水平，干预重要结合位点，阐明p63-增强子在基因表达和细胞表型的调控机制。为改善上皮病变提供新靶点和理论基础。

Abnormal repairing and remodeling in nasal epithelium cause the initiation and development of nasal mucosal inflammation. The applicant has revealed the important roles of epithelial transcription factor (p63) in contributing to the reduced repairing potential and aberrant remodeling in chronic nasal inflammation (published in Thorax; JACI). We have on the first time established the nasal epithelial stem cell culture system, and found that epithelial stem cells presented a reduced proliferation dynamic and incorrect differentiation cell fate in vitro (i.e., without the inflammatory microenvironment); and such evidence was similar to the pathological change in the epithelium in inflammatory tissue (i.e., in vivo change) (published in Sci Report; JCAI). However, the mechanisms of p63 in regulating nasal epithelial stem cell proliferation and differentiation are not clear. Our pilot study showed that during the proliferation stage, the epithelial stem cells derived from nasal polyps represented a lower transcriptional activity regulated by p63 and enhancer (a short region in chromatin DNA which promotes gene transcription),and such evidence was associated with the transcription levels of cell cycle and cell proliferation related genes. Therefore, we hypothesize that the regulatory mechanisms of the transcription factor p63 and the corresponding enhancers in nasal epithelial stem cells may be altered in the chronic inflammatory nasal mucosa, leading to the abnormal cell proliferation and differentiation. This project will utilize the human nasal epithelial stem cell system, use ChIP-seq technique to analyze the chromatin DNA binding regions p63 and the corresponding enhancer regions, use RNA-seq techniques to analyze the gene expression profiles, and intervene the identified p63-enhancer loci to clarify the functional roles and mechanisms of p63 & enhancers in regulating gene expression and changes of cell proliferation/differentiation phenotypes.The results are expected to identify specific targets for the improvement of abnormal epithelial repairing and remodeling in nasal epithelium under chronic mucosal inflammation.