PKA在人iPS细胞分化为血小板中的作用及机制研究

输注血小板是预防和治疗血小板数量或功能障碍导致出血的不可替代的治疗方法。然而由于献血者缺乏和血小板保存损伤的存在，临床血小板始终供不应求。通过诱导多能干（iPS）细胞体外分化生产血小板可提供新来源。凋亡在血小板生成和保存中起重要作用，研究报道蛋白激酶A（Protein kinase A，PKA）通过凋亡调控血小板的寿命和存活时间。PKA是否促进iPS细胞体外分化、巨核细胞成熟和血小板产生尚无证据确认。我们前期建立hiPSCs无血清无支持细胞单层贴壁培养诱导分化血小板的方法，本课题拟通过构建PKA基因修饰hiPSCs，诱导分化巨核细胞和血小板，解析PKA在血小板生成中的作用及机制，为人外周血源iPSCs分化产生血小板的临床应用奠定基础。

At present, platelet transfusion is still an irreplaceable treatment for preventing and treating bleeding caused by thrombocytopenia or platelet dysfunction. However, due to the lack of voluntary blood donors and the existence of platelet storage lesion, platelet shelf time is short, which makes the clinical platelet is always in demand. Induced pluripotent stem cells provide a new source for clinical platelet supply by differentiation and generation platelets in vitro. Apoptosis plays an important role in the process of platelet generation and preservation. It is reported that protein kinase A (PKA) determines platelet life span and survival by regulating apoptosis. There is no evidence to confirm whether PKA promotes iPS cells differentiation，the maturation of megakaryocytes and platelet production. We have established a feeder-free adherent monolayer differentiation culture protocol in serum-free medium with sequential addition of growth factors for hiPSCs differentiation to megakaryocytes and platelets in our previous work. This project is intended to modify the human peripheral blood derived iPS cells by overexpression or knockout PKA to investigate the effect of PKA on hiPSCs differentiation to MKs and platelets. We plan to add PKA activator or inhibitor to culture medium during normal hiPSCs differentiation to appreciate the regulatory mechanisms of PKA at each phase of a developmental process，such as iPSCs differentiation to MKs, MKs maturation and platelet production. We hope we can elucidate the effect and mechanism of PKA on platelet generation and storage, which can accomplish clinic scale platelet production in the end.