Investigation Of The Mechanism In Vitro Underlying The Effect Of Decitabine On The Treatment Of Thrombocytopenia After Transplantation

Background and Objective:Refractory thrombocytopenia after transplantation severely affects patient's survival.Its pathogenesis may be related to abnormal immune conditions and impaired bone marrow microenvironment.Studies have shown that low-dose decitabine can alleviate refractory thrombocytopenia,however,the mechanism is still unknown.Megakaryocyte reconstitution maybe associated with ROS in the bone marrow microenvironment,and decitabine can affect megakaryocyte reconstitution by altering ROS expression levels.This study will play an important role in the diagnosis and treatment of thrombocytopenia.Methods:1?Umbilical cord blood stem cells were selected and primary megakaryocyte culture system was constructed in vitro.Flow cytometry was used to detect the expression of CD42+ cells,platelet release,polyploidy of megakaryocytes,and changes of ROS during the culture of primary megakaryocytes.2?In order to explore the effects of decitabine on the differentiation and maturation of megakaryocytes,flow cytometry was used to analyze the expression ratio of CD41+CD42+ cells,ROS and platelet release when primary megakaryocytes were treated with different concentrations of decitabine(1n M?10n M?100n M).Then we change the action stage of decitabine and use flow cytometry to analyze ROS expression levels of primary megakaryocytes.Results:1?The mean proportion of CD42+cell was(29.35±0.82)% in the 12 th day of cell culture,and the mean proportion of CD42+ cell was(44.43±2.45)% in the 14 th day(p<0.05).2N,4N,8N,16 N megakaryocytes and platelets release were detected in the 14 th day of cell culture.The ROS is increasing during the culture of primary megakaryocytes(p<0.05).2?Cells were treated with different concentration of decitabine.The proportion of CD41+CD42+ cells in decitabine10 n M group was higher than that in the control group,decitabine1 n M group and decitabine100 n M group(p<0.05).The ROS of decitabine 10 n M group were higher than the control group,decitabine1 n M group and decitabine100 n M group(p<0.05).The platelet release was higher In decitabine10 n M group than the control group,decitabine1 n M group and decitabine100 n M group(p<0.05).In decitabine10 n M group,the proporation of CD41+CD42+ cells in the 14 th day were higher than the proporation in the 12 th day and the 13 nd day.To change the action stage of decitabine and finally we found that ROS is higher in the group which decitabine was added in the 7th day.Conclusion:We successfully established the primary megakaryocyte culture system.The proliferation,maturation and differentiation of megakaryocytes are accompanied by increasing ROS levels.Decitabine has a concentration-dependent effect on the differentiation and maturation of megakaryocytes.It can also increase ROS levels at a specific stage,and finally promote the differentiation and maturation of megakaryocytes.