湘西苗医刘氏小儿推拿流派“推五经”调节哮喘患儿免疫平衡的表观修饰机制

Asthma immunological pathogenesis is the disbalance of Th cell subsets-Th1, Th2, Th17 and Treg. Liu family of Miao medicine massage for child diseases in the west of Hunan Province is a famous massage school in China. The primary research has proved that the treatment of clinical catabasis must focus on regulating the immune balance to prevent recidivation. Histone acetylation plays an important role while gene-specific transcription factor T-bet, GATA3, RORγT and FoxP3 are regulating differentiation of Th cell subset. We speculate that five-meridian management with Tui-na massage is likely to regulate the immune balance through histone acetylation. The research attempts to take the above four Th cell subsets as object, which adopts the medical techniques such as real-time quantitative PCR (qRT-PCR), chromatin immunoprecipitation assay (ChIP-assay),western blotting and flow cytometry. It focuses on how five-meridian management with Tui-na massage dynamically influences the molecular mechanisms of histone acetylation modification of T-bet, GATA3, RORγT and FoxP3 gene promoter sub-region H3K9, H3K14, H3K18, H3K23, H4K5, H4K8, H4K12 and H4K16 histone acetylation. The research also focuses on how five-meridian management with Tui-na massage relates to relevant genes, differentiation of Th cell subsets, and clinical efficacy from dose-effect and time-effect. In order to provide scientific basis for the prevention and treatment of ashama, it will elementarily elucidate the regulation of immune balance from a new perspective of epigenetics.

哮喘的免疫学发病机制是Th细胞亚群（Th1、Th2、Th17、Treg）的失衡。湘西苗医刘氏小儿推拿是国内著名的小儿推拿流派。我们前期已证实本流派"推五经"能通过调节免疫平衡来防止儿童哮喘的复发。组蛋白乙酰化修饰在特异性转录因子T-bet、GATA3、RORγt及FoxP3调控Th细胞亚群的分化中发挥重要的作用。我们推测"推五经"可能通过组蛋白乙酰化修饰途径来调节哮喘患儿的免疫平衡。本研究将采用实时定量PCR、染色质免疫共沉淀、蛋白质印迹及流式细胞术等技术，从量效及时效关系动态研究"推五经"对T-bet、GATA3、RORγt及FoxP3基因启动子区域H3K9、H3K14、H3K18、H3K23、H4K5、H4K8、H4K12及H4K16乙酰化修饰的影响及与相关基因、蛋白表达、Th细胞亚群分化、临床疗效之间的关系，从表观遗传学的新视角初步阐明其调节免疫平衡的机制，为防治哮喘提供科学依据。

哮喘的免疫学发病机制是Th 细胞亚群（Th1、Th2、Th17、Treg）的失衡。湘西苗医刘氏小儿推拿是国内著名的小儿推拿流派。本研究将采用实时定量PCR、染色质免疫共沉淀、蛋白质印迹及流式细胞术等技术，从量效及时效关系动态研究了（1）Th 细胞亚群（Th1、Th2、Th17、Treg）的水平;（2）研究了PBMC的水平；CD4+T细胞的百分数以及其中T-bet、GATA3、RORγt及FoxP3的mRNA表达水平和蛋白表达。（3）研究了CD4+T细胞T-bet、GATA3、RORγt及Foxp3基因启动子区域组蛋白H3、H4 各位点乙酰化状态。结果显示，苗医刘氏小儿推拿“推五经”对Th 细胞亚群有明显下调。CD4+T细胞的水平得到改善。对T-bet、GATA3、RORγt及FoxP3的mRNA表达水平和蛋白表达有明显的影响。T-bet、GATA3及FoxP3的启动子区域组蛋白H3乙酰化状态的H3K9Ac变化也比较明显。证明了“推五经”对H3K9乙酰化修饰的影响及与相关基因、蛋白表达、Th 细胞亚群分化均有恢复作用。说明了哮喘的免疫性炎症得到了有效的改善。揭示了“推五经”对调节哮喘患儿免疫平衡的表观修饰机制。从表观遗传学的新视角初步阐明其调节免疫平衡的机制，为防治哮喘提供科学依据。本项目后续还将通过更大样本，更加深入的探讨“推五经”作用的分子机制，完善其作用机理，为防治儿童哮喘做出贡献。