利用Microfluidic系统研究血流速度对巨核细胞生成血小板的信号调控机制

Megakaryocyte (MKs) are exposed to shear flow as they migrate from the bone marrow hematopoietic compartment into circulation to release pro/preplatelets into circulation blood. Less mature MKs may enter circulation and mature under shear flow. Mechanotransduction is much less understood in MKs, especially in this period for platelet release. In our previous experiments, we found that the MKs ploidy was affected by shear flow. In this study, we plan to use fetal liver derived megakaryocyte at different mature combined with Microfluidic detection to elucidate the specific transcriptional changes that occur in MKs following exposure to shear flow, within different flow rate and affected time. MKs ploidy, DNA maturation and platelet biogenesis will be detected using flow cytometry and in situ confocal microscopy. Immunoblot and phosphorylation analysis evaluation for MAPK pathways ERK1/2、p38、JNK、ERK5, Inhibitor influence for this process. Intravital Confocal Microscopy was used to observe the process for platelet produce from MKs. All these results are contributed to elucidate the MAPK pathways transduction during MKs produce platelet and mature under shear flow, will provide the theoretical basis for thrombocytopenia mechanism.

巨核细胞在骨髓分化成熟后需要穿过血窦屏障在血管内血流压力作用下释放血小板，部分未成熟巨核细胞还可释放到血流中完成成熟分化过程。压力信号传递在血流作用下巨核细胞释放血小板及自身成熟的信号传递机制研究甚少。我们在前期实验发现血流压力作用可加快巨核细胞多倍体形成。MAPK作为主要的压力信号传递通路，其参与该过程的信号传递机制尚不明确。本研究拟采用胎肝细胞诱导生成巨核细胞结合Microfluidic技术研究不同血流压力作用下（流速和时间）MAPK信号通路在巨核细胞生成血小板及自身成熟过程中的作用机制，观察一系列巨核细胞成熟指数以及血小板相关指标；免疫印迹和磷酸化蛋白分析方法确定MAPK家族ERK1/2、p38、JNK、ERK5等蛋白组份的活化状态及其相关上下游作用分子；抑制剂对信号传递的作用以及活体共聚焦显微镜技术动态观察巨核细胞生成血小板的过程，从而为血小板数量异常性疾病的发病机制提供理论基础。

巨核细胞在骨髓中并不生成血小板，只有在穿过nich的时候才会形成前血小板，现有研究表明，成熟的巨核细胞释放入外周血后在肺生成血小板，所生成的血小板约占人体血小板数量的30%左右，研究显示压力信号传递在血流作用下巨核细胞释放血小板及自身成熟过程具有重要的作用。。本课题聚焦血流压力对巨核细胞MAPK信号传递通路在血小板生成中的作用机制研究，我们分别对巨核细胞在不同血流速度作用下成熟度进行分析，并观察在内皮细胞存在的情况下对巨核细胞成熟度及血小板生成情况进行检测，利用microfluidic系统，体外诱导培养的不同成熟度巨核细胞分别检测在不同血流速度作用下多倍体形成情况和前血小板生成情况分析，同时收集不同天数的巨核细胞进行蛋白质谱分析，分析获得MAPK信号通路中的相关蛋白表达信息，为血流压力在巨核细胞生成血小板过程中的MAPK相关信号蛋白表达谱的改变提供了实验证据。