Study On The Effects And Mechanisms Of ABA,High Lipid Plasma And PKA In The Differentiation Of Human IPSC Into Megakaryocyte

Part One:Abscisic Acid Stimulates Megakaryocyte Differentiation from Human iPSC in VitroIn clinical,the supply of platelet is often difficult to meet the needs of blood transfusion.To solve this problem,it is imperative to establish and optimize the method of differentiating functional platelets from CD34~+cell or human pluripotent stem cell(h PSC).We found that abscisic acid,ABA)can increase the production of megakaryocyte(MK)and platelet induced by human PSC(h PSC).In the process of platelet production,ABA treatment not only promoted the production of CD41~+MK on day 14,but also increased the production of CD41~+/CD42a~+,CD41~+/CD42b~+MK and platelet on day 19.Among them,ABA played a more important role during 0-14 days.In addition,the combination of ABA and its receptors LANCL2 and GRP78 mediated PKA cascaded activation of Akt and ERK1/2 signaling pathways,which played an important role in MK maturation and platelet production.Part Two:Role and Mechanism of High Lipid Plasma in Differentiation of Human iPSC into Megakaryocyte in VitroClinically,it is found that the incidence of stroke in patients with hyperlipidemia is much higher than that in non-hyperlipidemia,and hyperlipidemia is related to high platelet count,which suggests that hyperlipidemia may be related to platelet formation.Therefore,from September 2010 to December 2019,we statistically analyzed 317 patients with cerebral infarction in the Department of Neurology,First Affiliated Hospital of PLA Naval Military Medical University,and 381 healthy or individually diagnosed with hyperlipidemia in physical examination,and studied the influence of blood lipid on platelet,red blood cell and white blood cell count in the two groups.The results showed that hypertriglyceridemia was related to the formation of granulocyte-megakaryocyte cell line,and hypercholesterolemia was related to the formation of megakaryocyte line in healthy people.Hyperlipidemia is also associated with the increase of platelet level in patients with cerebral infarction.Adding different concentrations of plasma to APEL can promote the differentiation of human iPSC into hematopoietic-MK in vitro.The research shows that lipid components in high-fat plasma can promote the differentiation of human iPSC into hematopoietic-MK in vitro,which may promote the activation level of TPO-mediated JAK2,AKT,ERK1/2(MAPK1/2)and STAT3 signaling pathways through fatty acid transfer by fatty acid transferase CD36,and participate in the differentiation process of MK in vitro.Part Three:Preliminary Study on The Role of PKA in Differentiation of iPSC into Megakaryocyte in VitroProtein kinase A(PKA),as the most important effector of c AMP signaling pathway in platelets,can phosphorylate many proteins activated by platelets.PKA can regulate platelet apoptosis,and affect platelet life and survival time in vivo and in vitro.At present,there is no evidence that PKA can influence the differentiation process of human iPSC into MK and platelet.In this part of the study,we constructed human peripheral blood i PS cell lines with PKA overexpression and knockout.At the same time,our preliminary results show that PKA knockout can promote the differentiation of human iPSC into MK in vitro.