Effects Of Bone Marrow Mesenchymal Cells From Immune Thrombocytopenia Patients On The Biological Behaviors Of Megakaryocytes

Objectives Bone marrow mesenchymal cells(BMCs)from both Normal controls and immune thrombocytopenia(ITP)patients were studied in our research,meanwhile the megakaryocytic differentiation and BMCs-Megakaryocytes co-culture models were also established.Changes in the survival,proliferation and cytokines expression of BMCs from ITP patients as well as their influences on the differentiation and apoptosis of megakaryocytes under in vitro co-culture conditions were investigated.We want to identify deficiencies of BMCs derived from ITP patients,so as to deepen our understanding of the underlying mechanisms as well as to provide a new therapeutic target for better treatments of ITP.Methods Bone marrow samples were collected from 7 ITP patients and 5 normal controls,and BMCs were cultivated by the whole marrow adherent method.The surface markers of BMCs(CD105、CD90、CD73、CD34、CD45、CD19、CD11b、HLA-DR)and the basal cell apoptosis rate were analyzed by Flow cytometry(FCM).Proliferation of BMCs were assessed by Cell Counting Kit-8(CCK-8)method.The expression levels of IL-6、IL-11、TPO、SCF mRNA and protein in BMCs were detected by Real-time Fluorescent quantitative PCR(RT-qPCR)and Enzyme-linked immunosorbent assay(ELISA)respectively.Phorbol 12-myristate 13-acetate(PMA)was used to stimulate megakaryocytic differentiation in HEL cells.The induced cells were collected for morphological observation and surface proteins CD41 a 、 CD42 b detection using FCM.Mitomycin C was used to inhibiting BMCs proliferation to prepare feeder cells.The induced HEL cells were grouped and co-cultured with feeder cells from either normal controls or ITP patients,followed by surface protein CD41a、CD42b detection and apoptosis analysis through FCMResults We found that BMCs from ITP patients were larger in appearances,grew progressively slowly,and cell basal apoptotic rates were higher than that of normal controls.In addition,BMCs from ITP patients also showed defects in expressing cytokines IL-6 and SCF.Our results also demonstrated that HEL cells showed a series of characteristics referring megakaryocytic differentiation when treated with 5ng/ml PMA,including the increase in cell size、nuclear lobulation and expression of surface protein CD41 a,and the CD41 a positive rate was more than 90% after 72 h.Whereas,there was no obvious CD42 b expression on HEL before and after PMA treatment,indicating that the induced HEL cells were in the early differentiation stage of megakaryocytic development.BMCs from normal controls significantly reduced the apoptosis rate as well as promoted the CD41 a expression of induced HEL through co-culture in virto.Whereas,this capability were much weaker in ITP BMCs.Conclusion BMCs from ITP patients exhibited aberrant morphology,impaired proliferation and excessive apoptosis compared with cells from normal controls.In addition,they also showed defects in expressing some of the cytokines that related to the megakaryocytic developments.ITP BMCs also exhibited limited capability in supporting megakaryocytic differentiation and survival under co-culture conditions,which may be correlated with the aforementioned deficiencies in them.The dysfunction of BMCs in ITP patients may contribute to the impaired platelet production in them.