从Th17/Treg失衡和Notch通路探讨益气养阴清热化瘀方调控ITP血小板破坏及生成的机制

Pathogenesis of immune thrombocytopenia is mainly related to the following factors: a loss of tolerance to platelet antigens and a phenotype of accelerated platelet destruction and impaired platelet production. Increasing number of studies found that CD4+Th subset especially Th17 cells and Treg cell subsets balance disturbance play an important role in the pathogenesis of ITP; And Notch signaling pathways plays a key role in the hematopoietic stem cell differentiation, and the megakaryocyte maturation process. Based on the hypothesis, in this study the rat model of ITP as the carrier, We will use the FCM, Real-time Quantity PCR, Western Blot and other technology to detect Th17/Treg and their differentiation pathway related transcription factor and cytokine expression, and the relationship among the receptors, ligands and linear analysis of indicators of Notch family,and the relationship between the platelet destruction and production.trying to resolve its role in the onset of immune thrombocytopenia, Interpret that whether the efficacy Yiqiyangyin Qingre Huayu Recipe and its decomposed can regulate the imbalance of Th17/Treg, the Notch signal pathway, thereby inhibiting platelet destruction, promoting platelet production to play immunomodulatory effects, in order to provide the scientific experimental basis for the treatment of ITP.

免疫性血小板减少症（ITP）发病主要与以下因素有关：血小板表面抗原耐受缺失、血小板破坏加速以及血小板生成受损。越来越多的研究发现，CD4+Th细胞亚群尤其是Th17细胞和Treg细胞亚群的平衡紊乱在其发病过程中起重要作用；而Notch信号通路在造血干细胞分化、巨核细胞发育成熟过程中起关键作用。基于假说，本研究以ITP模型鼠为载体，采用FCM、 Real-time Quantity PCR、Western Blot等技术检测Th17/Treg及其分化途径相关转录因子及细胞因子表达以及Notch家族受体、配体，线性分析指标间关系以及与血小板破坏/生成的关系，试图解析其在免疫性血小板减少症中的发病作用，阐释确有疗效的益气养阴清热化瘀方及其拆方是否调节Th17/Treg失衡，调控Notch信号通路，进而抑制血小板的破坏，促进血小板生成发挥免疫调节作用，以期为其治疗ITP提供科学的实验依据。

本项目以免疫性血小板减少症（ITP）的免疫失衡机制中T细胞分化异常为研究重点，选用临床验方“益气养阴清热化瘀方”为干预药物，并设立高、中、低剂量及本方的3个拆方组，进行了一系列药效、机制的研究：（1）制备益气养阴清热化瘀方并进行质量评价（2）建立ITP小鼠模型（3）对益气养阴清热化瘀方合方及拆方治疗ITP模型进行药效学评价（4）研究本方调节ITP模型Th17/Treg 分化失衡的作用及分子机制（5）探索本方对ITP模型Notch 信号通路影响的分子机制（6）线性分析ITP 鼠Th17/Treg 失衡与Notch 信号通路以及与血小板生成/破坏的关系。得出以下结果：（1）优化了ITP小鼠模型，成模率高，且符合ITP的发病机制（2）验证了本方具有提升ITP模型小鼠外周血血小板计数（与模型组比较P＜0.01），并改善骨髓巨核细胞的产板能力的药效学作用（3）通过流式细胞术、ELISA、Q-PCR、Western-Blot等手段，检测Th17/Treg分化相关蛋白/细胞因子，发现本方能够下调ITP模型STAT3、STAT5、RORγt蛋白/基因的表达，上调FoxP3的蛋白/基因的表达；恢复Th17/Treg分化平衡，增加Treg细胞的数量和百分比，并增加其功能活性，上调TGF-β、IL-10细胞因子的分泌；减少Th17细胞的分化，并抑制下游炎症因子IL-17、IL-21的释放，最终达到调节免疫，发挥对ITP的治疗作用（4）发现本方能有效抑制Notch通路受体Notch1-4和Notch通路配体Jagged1-2的异常激活，从而影响T细胞的分化。验证了前期的假说，同时发现中药合方高、中、低剂量组无论从药效学还是分子生物学上其作用效果及干预广度均优于拆方各组，再次证明了中药复方对于调节免疫存在着多靶点、多途径，协同作用的优势，为临床运用本方治疗ITP提供了科学的实验依据。