靛玉红调控Tregs和巨核细胞治疗ITP的实验研究

It has been discovered that ITP patients have aberrant regulatory T cells and megakaryocyte maturation. Our previous studies showed that reduced amount and functions of regulatory T cells and impaired megakaryocyte maturation were involved in ITP. Increasing the amount and functions of regulatory T cells can provide effective therapeutic strategies for this disease. Our studies found that indirubin effectively decreased platelet destruction by lowering CD4+ T cells and increasing regulatory T cells relative count. We also observed that CTL and platelet-specific antibodies could induce platelet destruction and impaired megakaryocyte maturation. Regulatory T cells suppressed the activation and proliferation of CTL and auto-reactive B cells and prevented their effects on megakaryocyte maturation, resulting in elevated platelet number. Based on these reports, we firstly speculate that indirubin could reduce platelet destruction and promote platelet production via modulating regulatory T cells and megakaryocyte. To test this hypothesis, we intend to use an active model of chronic ITP mice and in vitro culture system of regulatory T cells and megakaryocyte as targets, trying to find the effect of indirubin in modulating regulatory T cells and megakaryocyte in ITP and its possible mechanisms.

ITP患者存在调节性T细胞异常和巨核细胞成熟障碍。课题组前期研究发现ITP患者Tregs数量和功能显著降低，巨核细胞成熟障碍，调控Tregs数量和功能、促进巨核细胞成熟可增加ITP血小板数量。靛玉红可通过提升Tregs数量，有效改善ITP血小板减少。后续研究发现CTL和血小板特异性抗体不仅能够导致血小板破坏增多，还能抑制巨核细胞成熟和凋亡，减少血小板生成。Tregs能够抑制CTL和自身反应性B细胞的活化增殖，减少血小板破坏，阻断其对巨核细胞成熟的抑制作用。基于这些研究，我们提出靛玉红通过调控Tregs数量和功能、巨核细胞成熟和凋亡，减少血小板破坏，促进血小板生成，提升ITP血小板数量这一假说。为验证该假说，我们拟构建主动型慢性ITP小鼠模型，体内探讨靛玉红对主动型慢性ITP小鼠的治疗作用；体外研究靛玉红对Tregs数量和功能、巨核细胞成熟的调控机制，明确靛玉红在ITP治疗中的作用及机制。

免疫性血小板减少症（ITP）是一种临床最常见的出血性自身免疫性疾病，免疫耐受失衡以及巨核细胞成熟障碍从而导致血小板破坏增多、生成减少是ITP重要的发病机制，其中T细胞稳态失衡是免疫耐受失衡的重要原因。靛玉红是中药青黛的有效成分，具有止血、提升血小板的作用，而目前其作用机制不明。我们的研究发现靛玉红可以提升Tregs的数量和功能，诱导自身反应性T细胞凋亡，提高CD4+T细胞PD1的表达，作用于PD1/PTEN/mTOR通路促进T细胞自噬从而维持T细胞的稳态。同时靛玉红对巨核细胞的作用具有浓度依赖性，低浓度的靛玉红(50ng/ml)可以促进巨核细胞分化、发育、成熟及血小板释放，而高浓度(5μg/mL)的靛玉红能够诱导未成熟巨核细胞发生无效凋亡，该部分巨核细胞凋亡后并不生成血小板。我们通过主动型慢性ITP小鼠模型中验证靛玉红可以通过上调Tregs细胞的数量和功能从而提升血小板，对ITP小鼠模型可以起到治疗作用。我们的研究发现靛玉红可以干预血小板破坏和生成两个方面，共同促进血小板数量的提升，为靛玉红治疗ITP提供了理论依据。