Effects Of Bone Marrow Mesenchymal Stem Cells On The Proliferation, Apoptosis And Imatinib Sensitivity Of K562 CML Cells

ObjectiveChronic Myeloid leukemia(CML)is a hematopoietic stem cell disease that is characterized by the existence of the Philadelphia chromosome(Ph)and the Bcr-Abl fusion oncogene,which translates into a kind of protein bear strong tyrosine kinase activity.Although the clinical application of tyrosine kinase inhibitor(TKI)make great progress,it also bring another severe problem—drug resistance.There are a lot of evidences show hematopoietic stem cells localize to specialized microenvironmental niches in the bone marrow(BM)which provide critical signals to regulate hematopoietic stem cells numbers and quiescence,and support its preservation.What’s more,bone marrow microenvironment is closely related with tumor progression,progression and drug resistance.As one of the most important stromal cells,mesenchymal stem cells(MSCs)play a major role in this process.Thus,we compare the change in CML cells before and after culture with bone marrow mesenchymal stem cells(BM-MSCs)in vivo and vitro,aim to investigate the effect of BM-MSCs on CML cells and provide the basic for resitricting TKI resistance.MethodBM-MSCs were obtained from BM of patients with CML.BM-MSCs were separated with the combination of gradient centrifugation and adherent screening method,and identified by morphological characters.CML cells proliferation curves were drawn.Cell cycle were determind by flow cytometry.Also,we established a subcutaneous xenotransplanted model of CML cells and CML cells + BM-MSCs in BALB/C nude mice,treating them with or without Imatinib(IM).Tumor volume was measured,and tumor inhibitory rate was calculated.We detected apoptosis with TUNEL and H-E method.The protein expression of Bax、Bcl-2、Caspase-3 and in Jak/Stat5 pathway were tested by immunofluorescence and Western Blot method.Result1.In the first generation,BM-MSCs were in large shape with prominecies.After 2-3 times cell generations,BM-MSCs were charactered with typical features.2.The proliferation of K562 cells was slower after co-cultured with BM-MSC.Campared with the control group,volume of implanted tumor in K562 cells and BM-MSCs was decreased from 2.63±0.06 to 1.97±0.04(P<0.05).3.Detected the cell cycle of K562 cells which were co-cultured with BM-MSCs in different concentrations(100:1、10:1、5:1、1:1)in vitro,indicating that BM-MSC inhibited K562 cells in G0/G1.In vivo,apoptosis in K562 cells implanted tumor decreased which were injected with BM-MSCs(P<0.05).The expression of Bax、Caspase-3 decreased,while Bcl-2 protein increased(P<0.05).4.Compared to the K562 + IM group,apoptosis in K562 +BM-MSCs + IM group increased(P<0.05).The expression of Bax、Caspase-3 was higher in K562 +BM-MSCs + IM group,while Bcl-2 protein decreased(P<0.05).5.Detected the protein expression in IL-7R/Jak/Stat5 pathway with immunofluorescence and Western Blot method.Result showed that IL-7R、Jak-1、 Jak-3、Stat5、p Jak-1、p Jak-3、p Stat5 in K562 +BM-MSCs + IM group expressed higher than the control group.Conclusion1.BM-MSCs isolated from BM in CML patients inhibit proliferation and apoptosis of CML cells.And,BM-MSCs stop CML cells at G0/G1,maintain them in a life-long living condition.2.BM-MSCs isolated from BM in CML patients protected CML cells from the attack of IM.3.IL-7R/Jak/Stat5 pathway is probably involved in this process of BM-MSCs decreasing the IM sensitivity CML cells.