CGRP通过甲基化调控骨髓间充质干细胞维持骨稳态的机制研究

Significant post-operative osteoporosis is observed in autografts for jaw bone reconstruction, and the mechanism of “aging”stem cells in such unbalanced bone homeostasisis remains obscure. Our published data and preliminary results demonstrate that innervation can prevent post-operative osteoporosis of bone grafts, and neuropeptides such as CGRP regulate mobilization and differentiation of BMMSC in bone regeneration. It has been reported that bone homeostasis correlates with WNT pathway in BMMSC. However, it is unclear whether and how CGRP regulates bone homeostasis. Based on these observations, we hypothesized that CGRP would prevent BMMSC aging to maintain bone homeostasis via regulation of DNA methylation in target genes such as WNT. The present study will clarify the role of CGRP in regulating BMMSC by using clinical samples from innervated bone grafts, rat models of mandibular denervation, CGRP knockout rats and the culture of BMMSC. We will observe the efficacy of CGRP in promoting osteogenesis differentiation and self-renewal in BMMSC, and clarify how CGRP controls EZH2 via p38 MAPK, thereby regulating DNA methylation in target genes such as WNT through H3K27me3. We believe our study will provide a novel insight of the neural microenvironment-based stem cell mechanism in bone homeostasis, which is important to improve functional reconstructions for major defects in jaw bones.

自体骨移植修复颌骨缺损术后会出现疏松化，这种骨稳态失衡的干细胞“老化”机制不明。我们的前期研究发现重建神经支配能预防移植骨的疏松化，且CGRP等神经肽在颌骨再生中影响BMMSC的动员和分化；骨稳态与BMMSC的WNT通路有关，但CGRP调控骨稳态的干细胞机制不明。据此提出假说：CGRP通过甲基化调控WNT等靶基因避免BMMSC老化，从而维持骨稳态。我们拟从个体、细胞及分子水平明确CGRP作用于BMMSC并维持骨稳态的机制：通过神经化移植骨临床标本、下颌骨失神经模型、CGRP基因敲除大鼠及BMMSC培养鉴定等，观察CGRP增强BMMSC成骨分化和自我更新能力的效果，揭示CGRP通过p38 MAPK控制甲基化酶EZH2，并经由组蛋白H3K27me3等调控WNT等干细胞相关基因的甲基化修饰机制。这将从神经微环境的新视点探讨骨稳态维持的干细胞机理，为改进颌骨大型缺损的功能性重建提供新思路。

本研究目的是解决自体骨移植重建颌骨术后的疏松化，阐明导致骨稳态失衡的干细胞“老化”机制。基于申请人主持的同期神经支配预防移植骨疏松化的临床试验，我们从个体、细胞及分子等水平，通过应用神经化移植骨临床标本、失神经动物模型以及BMMSC原代培养鉴定等，证实了临床骨移植标本及失神经动物模型内CGRP与BMMSC特性及骨稳态的相关性，明确了CGRP影响BMMSC成骨分化和自我更新能力的作用，初步阐明了CGRP增强BMMSC干细胞活性及与p38 MAPK通路的关系，发现EZH2及神经递质受体的异常甲基化表达。结论：CGRP通过p38通路及甲基化机制增强BMMSC干细胞活力，进而维持骨稳态。这从神经微环境的新视点探讨骨稳态维持的干细胞机理，为改进颌骨大型缺损的功能性重建提供新思路。