TNF-α通过GCN5介导的表观遗传修饰导致喉黏膜间充质干细胞功能异常的机制研究

Refractory of chronic laryngitis is due to the disturbance of homeostasis. Our research group have proved that laryngeal mesenchymal stem cells (LMSCs) resided in laryngeal mucosa play importance role in tissue homeostasis. We found that long-term stimulation of inflammatory factors such as TNF-α induced persistent changes of proliferation and differentiation of MSCs, suggesting it is an important factor of recurrence of chronic laryngitis. However, the mechanism remains elusive. Chronic inflammation could cause abnormal epigenetic modification. We found that the levels of histone acetylation were abnormal in LMSCs derived from inflammatory laryngeal tissue. Moreover, TNF-α could significantly affect the expression of GCN5, an important acetyltransferase, suggesting that GCN5-mediated epigenetic modification leads to permanent dysfunction of LMSCs during chronic laryngitis. Therefore, our project is subjected to determine the levels of key modification of histone acetylation and expression of GCN5 complex, to investigate the function of GCN5 on MSCs differentiation and self-renew based on siRNA and lenti-virus techniques, to explore the key target genes regulated by GCN5 by CHIP-chip analysis, confirm the molecular mechanism that TNF-α regulating GCN5, and establish large-animal laryngitis treatment model. Our study is aimed to get further understanding of interaction between inflammatory niche and stem cell, explore specific epigenetic modification that regulating MSCs function, and provide basic experimental evidence for novel strategy for treatment of chronic inflammatory diseases.

慢性喉炎难以治愈是由于炎症导致的局部组织稳态破坏。本项目组证明喉黏膜间充质干细胞（LMSCs）通过自我更新、多向分化及免疫调控维持局部稳态平衡，与喉炎发生发展密切相关。同时发现TNF-α长期刺激导致MSCs功能不可逆的改变，提示其是喉炎迁延不愈的原因，但机制不清。慢性炎症可导致干细胞表观修饰异常，本项目组发现炎症LMSCs中组蛋白乙酰化水平改变，而TNF-α可影响乙酰转移酶GCN5的表达，推测GCN5介导的表观遗传学改变导致了炎症LMSCs功能持续下降。依此本项目拟明确正常与炎症组织LMSCs中GCN5及其修饰位点表达差异，利用GCN5慢病毒过表达/干扰技术明确其对于细胞的作用，利用CHIP-chip技术明确GCN5调控基因靶点，探讨TNF-α调控GCN5的信号调控机制，建立大动物喉炎体内治疗模型，为深入探索MSCs在炎症发生中的作用、表观遗传与疾病发生、慢性炎症治疗新策略提供实验依据

慢性炎症导致组织干细胞功能出现持久异常，其机制与表观遗传修饰的改变密切相关。课题组前期发现喉黏膜间充质干细胞（LMSCs）具有自我更新及多向分化潜能，并具有免疫调节功能，然而上述功能在慢性炎症刺激下会发生不可逆的改变；进一步发现炎症LMSCs中组蛋白乙酰化水平改变，而TNF-α可影响乙酰转移酶GCN5的表达，推测GCN5介导的表观遗传修饰的改变导致了炎症LMSCs功能的持久改变。通过本项目的研究，我们明确了炎症因子TNF-α对LMSCs 关键信号通路表观遗传修饰的影响，阐明了TNF-α导致LMSCs多种功能持续异常的分子信号途径。本项目明确了GCN5 表达下降在慢性喉炎LMSCs 功能异常中的关键作用，发现GCN5通过调节FASL表达影响LMSCs免疫调节功能，以及通过调节VEGF分泌水平影响细胞的血管化潜能；同时，发现慢性炎症通过影响GCN5表达抑制LMSCs成脂、成骨多向分化潜能，并进一步阐明了GCN5通过调节Wnt配体转录水平影响Wnt/β-catenin通路活性进而调节细胞多向分化潜能的分子机制，为深入理解炎症状态下干细胞功能异常奠定重要理论和实验基础。基于分子机制研究，本项目进一步成功利用调控GCN5 表达水平及表观遗传修饰状态的方式恢复炎症环境来源LMSCs的多向分化潜能及免疫调节功能，为调控疾病状态中MSCs的功能奠定重要实验基础，并为基于LMSCs 的慢性喉炎治疗提供新策略。本项目相关内容发表论文7篇（SCI期刊论文6篇），部分研究成果达到国际先进水平，培养博士研究生5名、硕士研究生2名，完成项目计划内容。