血小板微粒通过诱导巨噬细胞分化和极化参与形成上皮性卵巢癌腹膜免疫缺陷的研究

About 70% patients with epithelial ovarian cancer (EOC) present wide metastasis along surface of peritoneum. Its peritoneum exhibit characteristics of immunologic defect. Our previous studies had shown that abundant extra vascular platelets and tumor associated macrophages infiltrate in peritoneum nearby tumor implants and those platelets were attached to macrophages very closely. In the mean time, number of platelet in peripheral blood of EOC was higher significantly than those in patients with borderline and benign epithelial ovarian tumor(P< 0.05). Those results suggested that activated platelets were involved in progress of EOC. Platelet microparticles (PMPs) are one of granules secreted from platelet. One of characteristics of PMPs contained abundant miRNA molecule, it could regulate those target cells function or differentiation by transfer genetic information, such as miRNA, by adherence to or endocytosis by cells. Based on miRNA transfer between PMPs and target cell, we are going to study how PMPs could induce macrophage differentiation and polarization to construct peritoneum immunologic defect microenvironment, which is suitable to pave the road to EOC metastasis to peritoneum. Then, based on in vitro and in vivo system, it will be confirmed that those macrophages stimulated by PMPs could accelerate EOC metastasis to peritoneum. Our study will elucidate the mechanism of interaction of platelet and stromal cell in peritoneum and explore novel therapy on EOC.

70%的上皮性卵巢癌（EOC）表现为腹腔内的广泛转移，其腹膜为免疫缺陷微环境。我们研究发现EOC腹膜内浸润了丰富的血管外血小板和肿瘤相关巨噬细胞，两者密切粘附；同时EOC患者外周血血小板数目显著高于交界性和良性卵巢肿瘤患者（P<0.05），提示激活的血小板与EOC的进展有关。血小板微粒（PMP）是激活血小板释放的重要颗粒，其显著特征是含有miRNA分子，通过与靶细胞粘附或者内吞后，传递遗传信息，调控靶细胞的功能和分化。基于RNA传递遗传信息的模式，本课题将筛选PMPs优势表达的miRNA分子和LncRNA分子，研究PMPs对巨噬细胞的分化和极化的调节，参与形成腹膜免疫缺陷的微环境，为EOC转移到腹膜提供适宜的条件；然后在体外和体内实验验证PMPs作用后的单核巨噬细胞能够加速EOC转移到腹膜。通过本研究，阐述腹膜内血小板与基质细胞相互作用机制，探索新的EOC治疗模式。

上皮性卵巢癌EOC患者体内凝血系统激活，表现为高凝状态，甚至有1%的患者以静脉血栓为首发症状，同时EOC患者的外周血血小板数量显著增加。而且血小板的数量与EOC患者的肿瘤负荷以及腹腔内的转移灶呈正相关。肿瘤可以引起血小板数量增加，同时也引起血小板功能活化。然而到目前为止，EOC中血小板生物学行为的机制尚不清楚。.在本项目中，我们分析了对比分析了218例卵巢良性肿瘤患者、59例交界性卵巢肿瘤患者和171例EOC患者外周血小板计数，结果显示：EOC患者血小板显著增加，且EOC患者术前患者术前血小板数目的增多，与EOC转移部位、术前CA125水平及总生存期呈显著正相关。我们模拟EOC患者体内高凝环境，利用凝血酶刺激血小板，发现刺激后血小板释放微粒（Platelet microparticles，PMPs）增多。PMPs与卵巢癌细胞系SKOV3共培养结果显示：PMPs介导卵巢癌肿瘤细胞发生上皮-间质化改变（epithelial-mesenchymal transition，EMT），SKOV3细胞中EMT相关标志物的表达水平发生显著改变。 同时，PMPs显著促进SKOV3细胞增殖和迁移能力。且发生上述功能变化的SKOV3细胞系，可以通过外泌体调控巨噬细胞向M2型巨噬细胞分化。芯片检测分析发现，凝血酶激活后血小板释放PMPs在EOC中发挥调控作用，主要由miR-939介导。靶向该miRNA分子，我们在SKOV3中过表达miR-939，观察到EMT现象增强，而敲低miR-939表达，SKOV3发生EMT过程被逆转。同时我们发现，SKOV3细胞摄取PMPs是由卵巢癌细胞表面分子分泌型磷脂酶A2型IIA（sPLA2-IIa）所介导，敲低SKOV3细胞中sPLA2-IIa分子后，肿瘤细胞摄取PMPs能力显著降低。.我们的研究结果揭示，激活的血小板释放PMPs，在EOC进展中通过介导肿瘤细胞EMT，促进肿瘤细胞恶性进展，并调控巨噬细胞向促肿瘤表型分化。PMPs发挥功能的基础，是其中特异性富集的miR-939分子，而sPLA2-IIa分子，通过介导EOC细胞摄取PMPs，在血小板和肿瘤细胞的交叉对话中扮演者重要角色。综上，我们的研究结果阐明了血小板在EOC中的全新作用通路，为卵巢癌靶向治疗提供了新的靶点。