ADAM17介导血小板受体GPIba 胞外段水解在免疫性血小板减少症中的作用机制研究

Immune thrombocytopenia (ITP) is an immune-related disease characterized by accelerated platelet destruction. However, the mechanism underlying this disease is not very clear, leading to the limitation on its treatment.Thus, investigating the molecular mechanism of platelet destruction in ITP would enhance our understanding on the pathogenesis of ITP and provide a new target and strategy for the treatment. ADAM17-mediated ectodomain shedding of platelet receptor GPIba (a subunit of GPIb-IX-V) has been reported to be involved in platelet destruction. Increased ectodomain shedding of GPIba was observed in ITP, suggesting its involvement in the pathogenesis of ITP. In a previous study, we demonstrated that GPIb-IX-V binds to TRAF4 (TNF receptor associated factor 4) and TRAF4 binds to p47phox, a subunit of NADPH oxidase (Nox), the origin of ROS production, which regulates ADAM17 activation. Whether GPIb-IX-V/Nox/ROS is involved in the ectodomain shedding of GPIba via ADAM17 activation in ITP has not been reported previously. In this study, we propose to analyse the association of GPIba ectodomain shedding with ITP through clinical samples and investigate whether GPIb-IX-V/Nox/ROS is involved in the ectodomain shedding of GPIba in ITP through in vitro and in vivo (ITP mouse model) experiments, illuminating its role in the platelet destruction in ITP. If accomplished on schedule, this study could provide a new strategy for ITP treatment.

免疫性血小板减少症（ITP）是以血小板破坏为主要特征的免疫相关性疾病，但其机制尚未完全阐明，故其治疗策略局限。深入探索ITP中血小板破坏的机制，可拓宽对ITP发病机制的认识，为其治疗提供新靶点和策略。金属蛋白酶ADAM17通过水解血小板GPIb-IX-V中GPIba胞外段参与血小板破坏。ITP患者GPIba胞外段水解增加，提示其参与ITP发病。本课题组发现GPIb-IX-V与NADPH氧化酶（Nox）相互作用，Nox是活性氧（ROS）的主要来源，而ROS调节ADAM17活化。但GPIb-IX-V/Nox/ROS是否通过ADAM17参与ITP中GPIba胞外段水解未见报道。本项目拟用临床样本分析GPIba胞外段水解与ITP相关性、体外实验及ITP动物模型研究GPIb-IX-V/Nox/ROS是否参与GPIba胞外段水解，明确其在ITP血小板破坏中的作用。如期完成，可为ITP的治疗提供新思路。

免疫性血小板减少症（ITP）是一种免疫性疾病，特征是血小板数目降低，使病人带有出血倾向，严重时可危及生命。近些年虽然治疗效果有很大改善，但疗效仍不令人满意。为此，深入探索ITP的分子机制，将为临床上对ITP采用更具有针对性的治疗手段及评估药物的治疗效果提供思路和理论支持。.本课题针对ITP，主要研究：1）血小板受体GPIba水解在ITP发病中的作用；2）血小板凋亡在ITP中的作用及机制；3）Th9细胞在ITP中的表达及意义。重要结果如下：1. 新型检测GPIba水解方法的建立。使用两种不同荧光标记的分别抗GPIba胞外段和胞内段的抗体，通过流式细胞仪来分析胞外段抗体对于胞内段抗体的结合的相对值来反映GPIba水解程度。2. ITP中GPIba胞外段水解增加。利用新建立的方法，发现同对照相比，ITP治疗前GPIba水解程度显著升高，同时GPIba膜表达量显著降低，但GPIba水解程度和膜表达量在治疗后均恢复到正常水平。提示GPIba水解可能参与ITP发病。3. GPIba水解程度可作为评估ITP激素治疗效果的指标。ITP经激素治疗达到缓解后，GPIba水解恢复到正常水平的时间显著快于血小板数目的恢复，而对无效患者，GPIba水解均未恢复到正常水平。提示GPIba水解程度可优于血小板数目作为评估激素治疗疗效的指标。4. ITP血浆诱导正常血小板凋亡。将ITP血浆与正常血小板共培养，模拟ITP发病环境，发现ITP血浆可诱导血小板凋亡。5. ITP血浆诱导血小板凋亡相关蛋白的表达。ITP血浆处理后，血小板内Bcl-xL表达降低，Bax升高和caspase-3活化，提示血小板凋亡可能参与ITP发病，靶向Bcl-xL和Bax有可能成为ITP治疗的新靶点。6. ITP中Th9细胞表达升高。与对照相比，Th9和Th17细胞在ITP活动组中显著升高，在缓解组中恢复至正常。与此相符，调控Th9和Th17分化的PU.1、IRF4、BATF及ROR-γt mRNA在ITP中的表达也呈相同趋势。7. ITP中Th9和Th17之间呈正相关。在ITP中，Th9/IL-9、Th17/IL-17、Th9/Th17、IL-9/IL-17之间呈正相关，IRF4、BATF和ROR-γt mRNA两两之间也呈正相关。本研究表明ITP中Th9表达异常，其可能通过与Th17细胞相互作用，共同促进ITP的发病。