益气滋阴方调控CaN/NFAT信号通路诱导免疫性血小板减少症中调节性T细胞生成的机制研究

Immune thrombocytopenia (ITP)’s main pathogenesis is the excessive activation of immune system and the destruction of platelets, easily to become relapsed or refractory. Regulatory T cells (Treg) which inhibiting immune hyperfunction and alleviating the ITP, is a hotspot of the disease. The traditional Chinese medicine pathogenesis of this disease is associated with "Qi and Yin," so we create Qi-supplementing and Yin-nourishing prescription which having remarkable clinical effect. Prophase research confirmed that the recipe promoting the ratio of Treg and the expression of CaN and NFAT protein; and CaN/NFAT pathways is a crucial factor in the generation of induced Treg. So we put forward the hypothesis"Qi-supplementing and Yin-nourishing prescription generate induced regulatory T cells by regulating CaN/NFAT pathway in ITP". This project intends to observe the recipe’s effect on ITP models and the generation of induced Treg in vivo. Then we adopt a target gene silencing and overexpression, use specific blocker and stimulant, use magnetic bead separation technology, in the process of generating induced Treg, observe the influence of this recipe on CaN/NFAT pathway key factor expression and phosphorylation levels, Treg proportion and its immune phenotype, transcription factors and cytokines, immune suppression function. Clarify the specific mechanism of how Qi-supplementing and Yin-nourishing prescription generate induced Treg, provide new targets for the treatment of ITP.

免疫性血小板减少症（ITP）的主要病机是免疫系统过度激活后破坏血小板，易复发并成为难治。调节性T细胞（Treg）能抑制免疫亢进并缓解本病，在ITP中促进其数量及功能恢复是目前的研究热点。本病中医多责之“气阴”，我们以此创立益气滋阴方，临床疗效显著。前期研究证实该方在体内能提高Treg比例，上调诱导生成Treg关键通路CaN/NFAT的相关蛋白的表达。因此我们提出“益气滋阴方可能通过调控CaN/NFAT通路诱导生成Treg进而治疗ITP”这一假说。本项目在体内观察该方对ITP模型的影响以及对诱导生成Treg的作用。在体外采用靶基因沉默及过表达、阻断剂及激动剂运用、磁珠分选等技术，观察该方在诱导生成Treg过程中对CaN/NFAT通路关键因子表达及磷酸化水平，Treg比例及其免疫表型、转录因子、细胞因子、免疫抑制功能等的影响。阐明益气滋阴方诱导生成Treg的具体机制，为其治疗ITP提供新靶点。

本研究在前期研究基础上，采用抗血小板血清法建立ITP大鼠模型，通过一般状态观察、血及骨髓检查、流式细胞技术、RT-PCR、ELISA、Western blot等方法观测益气滋阴方干预下小鼠整体状态、血小板、巨核细胞、Treg比例、转录因子和细胞因子、钙调蛋白信号通路蛋白的动态变化，验证益气滋阴方对ITP模型的治疗效果以及对Treg及其相关信号通路的作用，探明益气滋阴方促进Treg生成从而治疗ITP的分子机制。结论如下： .第一，抗血小板血清法建立ITP大鼠模型的紫癜症状稳定，外周血血小板减少，骨髓巨核细胞减少，T细胞免疫亢进，调节性细胞数减少，是一种符合临床ITP疾病表现和发病机制的动物模型。.第二，益气滋阴方可以促进大鼠外周血的调节性T细胞增殖，增加TGF-β、IL-10等关键细胞因子和分泌，从而减少体内亢进的淋巴细胞对外周血血小板及骨髓巨核细胞的破坏，改善ITP大鼠的临床指标和整体状态。.第三，益气滋阴方可以通过激活钙调蛋白信号通路的CaN和NFAT2的表达水平，促进关键转录因子Foxp3的表达，引起调节性T细胞数量恢复，恢复自身的免疫耐受状态，从本质上提升血小板计数并改善疾病。.该课题的完成有助于进一步诠释CaN/NFAT2信号通路在促进Foxp3表达从而诱导Treg生成中并治疗ITP中发挥的作用，以及益气滋阴方在其中的干预作用。该课题的研究成果为益气滋阴方治疗ITP提供一个新的靶点，但目前的研究只是初步成果，该组方的组份分析及其关键效应物质、信号通路激活的深入机制均有待在后续进行进一步的研究。