The Function Of TPO/c-MPL Signaling Pathway In Acute Myeloid Leukemia Bone Marrow Microenvironment

BackgroundThe form and function of bone marrow microenvironment are the result of the commoneffect of a complex network syetem which composed by multicellular and multiple factors.Usually, it is a three-dimensional structure containing stromal cells as the main body, andsome adhesion molecules and growth chemokines. Stromal cells are consisted byosteoblasts, fibroblasts, endothelial cells and tissue cells. In such a long time，the researchesabout bone marrow microenvironment were only observed the changes of adhension andsecretory function in bone marrow stromal cells after it was co-cultured with leukemic cells invitro. In1978, Schofield identified the"niche" so as to describe the microenvironment ofhematopoietic stem cells, after that different techniques had been used in animals by mangyscholars to research the effection of hematopoietic cell niche in maintaining the physiologicalfunction of hematopoietic stem cells. Purton found that hematopoietic stem cells relied onosteoblasts and vascular endothelial cells to realizing the self-renewal and differentiation. Theosteoblast was the major component of osteoblast niche, it was confined on the surface ofbone marrow cavity and it playedan important role in storaging long-term culture hematopoietic stem cells andmaintaining its physiological functions. Affluent hematopoietic precursor cells usuallygathered around endosteal osteoblasts in hematopoietic recovery after injury and bonemarrow transplantation model. The osteoblast niche could support the long-term survival ofhematopoietic stem/progenitor cells and starting cells of long-term culture in common.The formation and development of acute myeloid leukemia are closely related to thebone marrow hematopoietic microenvironment. But how the leukemia bone marrowmicroenvironment formats and the mechanism of hematopoietic malignancy in leukemia havenot been fully understood. The osteoblastic niche is the place of self-renewal anddifferentiation of leukemia cells especially leukemic stem/progenitor cell in leukemiaaberrant hematopoietic microenvironment. Quiescent leukemic cells is localized on the inner surface of the bone cavity so as to avoid the injury which caused by physicochemical factors,and this factor eventually becomes the mechanism of primary drug resistance and the relapseof leukemia after chemotherapy.A article by Hiroki has demonstrated that c-MPL is highlyexpressed in hematopoietic stem cells which stated in G0and located with osteoblasts in bonemarrow.Hematopoietic stem cells arequiescenced in these niche in order to avoid theinfluence of physicochemical factors.It can enter in the cell cycle to maintenance the normalhematopoietic under the stress situations. A research by Yamazaki has found that TPO/c-MPL signaling pathway plays an important role in the self-renewal and the G0maintenance of hematopoietic stem cell in hematopoietic stem cell niches. Blocking the TPO/c-MPL pathway can significantly reduce the number of hematopoietic stem cells which are inG0state in long-term culture.The interaction between thrombopoietin and its receptor c-MPL can promote theproliferation of megakaryocytic and increase the quantity of platelets in peripheral blood.Recent studies have shown that the osteoblasts in marrow strom secretes thrombopoietin, andc-MPL is not only expressed in different maturation stages of megakaryocyte, or earlyhematopoietic stem/progenitor cells, but also expressed in the primitive cells of acute myeloidleukemia, chronic myeloid leukemia and myelodysplastic syndrome patients.Because of the thrombopoietin has ecretion function in osteoblasts and the c-MPL ishighly expressed in AML cells. This experiment is supported by the National Natural ScienceFoundation, at first we detected the expression of TPO and its receptor c-MPL of nitial andrelapse acute myeloblastic leukemia patients to explore the relationship between theexpression levels and the relapse, refractory of leukemia;And then, successfully inducedbone marrow mesenchymal stem cells into osteoblasts, detecting the TPO secretion ofosteoblasts and c-MPL expression of AML cell lines HEL cells. In this experiment, theosteoblasts and acute myeloid leukemia cell line HEL cells were co-cultured in vitro in atwo-dimensional co-culture system in order to investigate the role of TPO/c-MPL signalingpathway in osteoblast niche which protecting the leukemia cells. We oserves the sensitivityof chemotherapeutic drugs and which to provide a new idea for the treatment of AMLrecurrence and resistance. Methods1．The expression of thrombopoietin and its receptor c-MPL（1）Collection of bone marrow mononuclear cells inAML patients and the normal.（2）The expression of c-MPL in AML patients and normal bone marrow mononuclearcells had been detected by flow cytometry.（3）The expression of TPO in bone marrow supernatant in AML patients and normalwere detected by ELISA.2．The secretion of TPO in osteoblasts which came from different source and theexpression of c-MPL in AML cell lines.（1）Induction of bone mesenchyme stem cells to osteoblasts and identification in AMLpatients and normal.（2）Cultivation of hFOB1.19cell and HEL cell in vitro.（3）The expression of c-MPL in HEL cell line was detected by flow cytometry.（4）The secretion of TPO in bone marrow cells，hFOB1.19cell and osteoblasts whichwere inducted from different source bone mesenchyme stem cells.3．The role of TPO/c-MPL signaling pathway in the protection of AML cells in bonemarrow microenvironment.（1）The two-dimensional co-culture system between HEL cells and osteoblasts wasbuilt.（2）Different the survival rate of HEL every group co-culture for24hours in vitro bydifferent concentrations of daunorubicin.Results1．c-MPL expression in bone marrow mononuclearcell cells are both more higher inrelatpse and primary patients of AML as compared to normal group(P<0.05); c-MPLexpression in relatpse group is significantly higher than primary group(P<0.05); theexpression of c-MPL can be cutted down after chemotherapy remission.2．The expression of TPO in bone supernatant of AML primary patients is higher thannormal(P<0.05), TPO expression in relatpse patients is more higher than primary patients.There is no siginificantly difference between chemotherapy remission of primary patients andnormal. 3． The cultivation, identification and induction to the osteoblasts of the bonemesenchyme cells of AML have been done. the Hfob1.19cell line and HEL cell line havebeen cultured in vitro.4．The expression of TPO is increasingly raised in the formation process of osteoblastswhich was induced by bone mesenchyme stem cells. The expression of induced osteoblasts inthe bone mesenchyme of AML is higher than the normal bone mesenchyme at the same phasepoint(P＜0.05).The osteoblasts line hFOB1.19can inject the TPO in vitro cultured.5．The expression of c-MPL in HEL cell line is8.32%, in DAMI cell line is6.10%.6．The curve of survival rate of HEL is horizontal S-shaped under the action of differentconcentration of daunorubicin each group that co-cultured for24hours in vitro,matchs thegeneral laws of the drug action. The cell viability of HEL+AML source of osteoblasts groupare highest by different concentration of DNR, cell survival rates of HEL cultured alone groupare lowest, and they are no significant difference between HEL+hFOB1.19group andHEL+normal source of osteoblasts.7． Calculating the IC50of daunorubicin by Spss13.0software. The IC50is（1.643±0.108）,（3.320±0.218）,（2.236±0.167）and（2.254±0.111）ug/ml of HEL aloneculture group, HEL+AML source of osteoblasts group, HEL+normal source of osteoblastsgroup and HEL+hFOB1.19group.The IC50of A group is significantly lower than the otherco-culture group(P＜0.05), group D is significantly higher than other groups(P＜0.05), groupB and group C have no significantly differences(P>0.05).Conclusion1．The highly expression of TPO and c-MPL have significantly hint meaning to thediagnosis of AML. The expression of TPO and c-MPL in AML relatpse patients are morehighly than primary patients. It indicates that the highly expression of TPO/c-MPL signalingpathway may be connected with the recurrence and refractoriness.2．The expression of TPO in osteoblasts which revulsived from AML bone marrowmesenchyme stem cells is more higher than osteoblasts which revulsived from normal bonemarrow mesenchyme stem cells and hFOB1.19cell line. The c-MPL is highly expressed inAML cells，especially in HEL cells.3．The osteoblasts and AML cells co-cultured in vitro have the function of protection against of the lethal effect caused by the chemo drug to the leukemia cells line.The role inprotection from the high expression of c-MPL in osteoblasts induced by the bonemesenchyme of AML is stronger.4．The above literatures suggest that the TPO/c-MPL signaling pathway may be the newtreatment target of the refractoriness’ relapse AML.