Beclin-1介导的自噬在原发性免疫性血小板减少症发病中的调控及分子机制研究

Primary immune thrombocytopenia(ITP) is an autoimmune mediated bleeding disorder in which platelet membrane proteins become antigenic and stimulate the immune system resulting in thrombocytopenia due to immune mediated platelet destruction and/or suppression of platelet production. Autophagy is a highly conserved lysosome-mediated catabolic process that allows cells to degrade to recycle nutrients and plays a primordial role in the regulation of innate and adaptive immune systems.There are several evidences that support the involvement of autophagy in autoimmune diseases. Our previous study confirmed that the protein levels of both Beclin 1 and LC3-II in active ITP patients were increased compared with the normal controls，while the expression of p62 was decreased significantly. Auto-antigens such as GPIIb/IIIa and/or GPIb/IX may derive from intracellular protein degradation as a result of autophagy, thus contributing to the initiation or perpetuation of the autoimmunity process in ITP. The function of autophagy in ITP is not clear, which requires more work. The present study aimed to explore the role of autophagy mediated by Beclin-1 in ITP using in-vivo and in –vivto methods. Thus we expect that future research on autophagy may lead to discover novel therapeutic targets offering novel opportunities to personalized medicine for the treatment of ITP.

T细胞对血小板自身抗原免疫失耐受是ITP免疫异常的重要机制。自噬作为一种高度保守的信号通路，参与固有和适应性免疫耐受调控，与自身免疫性疾病密切相关。前期研究证实ITP患者活动期T细胞Beclin 1及LC3-II表达明显升高，p62表达明显减少，Beclin 1介导的自噬活性在ITP患者体内增高，从而抑制淋巴细胞的凋亡，引起免疫系统对自身血小板表面抗原耐受丧失，产生抗体或致敏淋巴细胞破坏，导致血小板减少。本课题拟以血小板特异性抗原反应性T细胞-DC细胞共培养和ITP动物模型为切入点，以 Beclin 1、LC3-I、LC3-II及p62等关键因子为靶点，应用慢病毒构建Beclin 1基因沉默及过表达载体，验证疾病条件下Beclin 1介导的自噬通路蛋白的失衡在诱导自身抗体合成、细胞增殖、凋亡、耐受等功能紊乱及核心分子机制中的作用，为寻找干预ITP的新靶点提供理论和实验依据。

原发性免疫性血小板减少症（Primary immune thrombocytopenia，ITP）是临床上常见的血液系统疾病，是以血小板过度破坏和/或血小板生成减少为特点的自身免疫性出血性疾病，约占出血性疾病总数的1/3。众所周知，T细胞对血小板自身抗原免疫失耐受是ITP免疫异常的主要发病机制，深入研究ITP患者免疫失耐受机制，将有助于开辟恢复免疫耐受的干预新手段。新近研究表明，自噬通路在免疫耐受中发挥重要作用，自噬通路的紊乱很可能是诱发ITP的重要分子机制。前期研究证实ITP患者活动期T细胞Beclin 1及LC3-II表达明显升高，p62表达明显减少，Beclin 1介导的自噬活性在ITP患者体内增高，从而抑制淋巴细胞的凋亡，引起免疫系统对自身血小板表面抗原耐受丧失，产生抗体或致敏淋巴细胞破坏，导致血小板减少。我们研究结果显示GPIIb/IIIa反应性CD4+T细胞与DCs共孵育后，加入Beclin 1重组蛋白组LC3-I向LC3-II的转化增多，IFN-γ表达明显升高；加入Beclin 1抗体组LC3-II 表达未见明显降低，细胞凋亡活性降低，IFN-γ表达明显下降。ITP模型鼠骨髓巨核细胞Beclin 1及mTOR表达较对照组均升高。Beclin 1单抗组及慢病毒干扰载体组IFN-γ表达明显下降，IL-10的表达升高，Beclin 1重组蛋白组IFN-γ表达明显升高，而加入雷帕素实验组的小鼠较未采取任何干预措施的ITP小鼠IFN-γ表达明显降低，血小板数量较前改善。研究了Beclin-1介导的自噬在原发性免疫性血小板减少症疾病转归的影响，为揭示ITP患者免疫耐受的形成以及维持的机制、阐明ITP细胞因子紊乱现象与机制提供新的思路。