The Study On Neuropathy And Differentiation Of Neuron-like Cells In Bone Marrow Microenvironment In Patients With Acute Myeloid Leukemia

SECTION 1Neuropathy correlated with imbalanced Foxp3/lL-17 in bone marrow microenvironment of patients with acut myeloid leukemiaBackground:Acute myeloid leukemia(AML)is characterized by malignant clone of hematopoietic stem cells and accumulation of immature myeloblasts in bone marrow.Though the therapeutic strategy has made great progress,there are still many AML patients that fail to achieve complete remission(CR)and relapse at last.Chemotherapy resistance and immune system disorders are the main reasons.Therefore,it is of importance to clarify the pathogenesis of AML and explore novel therapeutic strategies.Bone marrow microenvironment,playing an important role in the development of leukemia,comprises a rich network of hematopoietic stem cells(HSCs),mesenchymal stem cells(MSCs),osteoblasts,adipocytes,sinusoidal vessels,perivascular reticular cells and bone marrow neural tissue.Bone marrow neural tissue,playing a key role in hematopoiesis and immunity,is composed of sympathetic nervous system(SNS)fibers,myelinating Schwann cells,nonmyelinating Schwann cells and nestin+ MSCs.Bone marrow hematopoiesis and differentiation are regulated by bone marrow neural tissue.Sympathetic nerve injury is closely related to the hematological diseases.Therefore,the damage of sympathetic nerve in AML bone marrow may be involved in the development of AML.Clarifying its effects and mechanisms is of importance for AML targeted therapy.Objectives:The aim of this study was to elucidate AML pathogenesis and find novel targeted therapy.We detected the expression of nerve-related factors[nestin,tyrosine hydroxylase(TH),GFAP and S100B]in BM of AML patients to explore the role of nerve injury in the development of AML.Furthermore,we assessed the prognostic impact of nerve-related factors expression levels and their association with the T helper-related molecules and clarified their clinical relevance.Methods:1.Patients and controls:A total of 100 patients with newly-diagnosed AML were enrolled in this study,and the control group consisted of 55 individuals.2.Treatment regimen and follow-up:The patients with non-M3 AML subtypes were treated with standard induction chemotherapy.The patients with acute promyelocytic leukemia(APL,subtype M3)received all-trans retinoic acid with or without concurrent induction chemotherapy.After the patients achieved CR,they underwent consolidation chemotherapy with a conventional dose of cytarabine and one anthracycline or with a high dose of cytarabine.The median follow-up duration was 15.5 months.3.Detemination of nerve-related molecules:To investigate whether nerve-related molecules(nestin,TH,GFAP and S100B)are involved in BM of AML patients,their expressions were examined using the immunohistochemical staining.For the immunohistochemical positively-expressed individuals,we further analyzed their expression levels.The expression of neurotransmitter Y(NPY)and norepinephrine(NE)in the bone marrow of AML patients and control group was detected by ELISA.4.Detemination of T helper-related molecules:To evaluate the immunological status in bone marrow microenvironment,Th-related molecules Foxp3 and IL-17 were detected using the immunohistochemical staining.Results:1.Nerve-related molecules were down-regulated in bone marrow of AML patients.2.The aberrant expression of T helper-related molecules was found in bone marrow of AML patients.3.The significant correlations were observed between nerve-and T helper-related molecules.4.No correlation was found between clinical characteristics and nerve-or T helper-related molecules in AML patients.5.Nerve-related molecules were associated with the survival of AML patients.Conclusion:1.The damage of sympathetic nerve in AML bone marrow is involved in the development of AML.2.Neuropathy togethered with imbalanced T helper immunology in bone marrow might play important roles in AML.SECTION 2The study on differentiation into neuron-like cells from bone marrow-derived mesenchymal tromal cells in patients with acut myeloid leukemiaBackground:BM-MSCs are a kind of multipotential stem cells that can differentiate into osteoblasts,adipocytes,neurons and various mature mesenchymal cells.BM-MSCs are key components of the hematopoietic microenvironment(HM),and regulate bone marrow hematopoiesis directly through a variety of cell adhesion molecules.Mature stromal cells derived from BM-MSCs not only provide mechanical support for hematopoietic stem cells(HSCs)to maintain normal hematopoiesis in the bone marrow,but also secrete a variety of cytokines to promote the differentiation of HSCs.Additionally,BM-MSCs participate in the occurrence and development of hematologic malignancies based on the characteristics of bidirectional immune regulation,multilineage differentiation and the homing of tumor.It was found that mice BM-MSCs inhibited the proliferation of leukemia cells and lymphoma cells in vitro and prevented the host rejection after allograft,which was beneficial to reconstruct normal hematopoiesis in vivo.However,some studies have found that BM-MSCs improved tumor proliferation and chemotherapy resistance.Other studies confirmed that BM-MSCs could promote the proliferation of leukemia cells,and protect leukemic cells from hemotherapy.BM-MSCs of patients with hematological malignancies have abnormal biological characteristics.For example,reduced proliferation,impaired differentiation,and aberrant expression of adhesion molecules were observed in AML-derived BM-MSCs.BM-MSCs of patients with aplastic anemia showed increased adipogenic differentiation and impaired osteogenic differentiation.Our previous studies have found that the nerve tissue in bone marrow is important for the normal hematopoiesis,and the neuropathy of bone marrow together with the imbalance of cellular immunity plays important roles in the development of AML.Normal BM-MSCs has the capacity to differentiate into neurons,while the neurons differentiation potential of AML BM-MSCs remains unknown.To explore the biological characteristics of AML-derived BM-MSCs and the ability to differentiate into neurons,we cultured and induced BM-MSCs derived from AML patients and controls in vitro,and then conducted a comparative study.Objectives:To understand the biological characteristics of BM-MSCs and the ability to differentiate into neurons in AML,we cultured and induced BM-MSCs in AML patients and controls in vitro,and then conducted a comparative study.Methods:1.Patients and controls:A total of 30 patients with newly-diagnosed AML were enrolled in this study,and the control group consisted of 18 individuals.2.Isolation and Expansion of BM Mesenchymal Stem Cells:Primary BM-MSCs were isolated by centrifugation using Ficoll density gradients.Mononuclear cells were seeded at 2×105 cells/cm2 and maintained in a humidified atmosphere with 5%CO2 at 37℃.After 24 hours,non-adherent cells were washed off and the medium was changed every 2-3 days.Adherent cells reached 80-90%confluences,and the cultures were by trypsinized and replated.BM-MSC cultures were assessed daily for changes in growth rates and morphology.Cell proliferation was measured using a cell counting kit-8.3.Identification of BM-MSCs:Imnunophenotype of MSCs was detemined by flow cytometry.The following monoclonal antibodies conjugated with FITC,PE or ECD were used for flow cytometric analysis:CD14,CD19,CD34,CD44,CD45,CD73,CD90,CD 105 and HLA-DR.Adipogenic differentiation was performed to differentiation assay.4.Induction of BM-MSCs:Both basic medium and inducer were added to the induction group,Basic medium was composed of L-DMEM supplemented with BDNF,NGF and 1%adjuvant N2.We prepared the following combinations of EGF,bFGF,BHA,and IBMX.The control group was treated with basic medium only.5.Detemination of nerve-related molecules:To investigate whether BM-MSCs differentiated into nurons,the expressions of nerve-related molecules(nestin,tubulin and GFAP)were examined using the immunohistochemical staining.Results:1.AML-derived BM-MSCs were successfully obtained.The morphology of BM-MSCs from AML patients was same as that of the control group.Compared with the control group,the number of primary AML-derived BM-MSCs grew more rapidly,with shorter doubling time.However,after subculture,we observed the decreasing proliferation of AML-derived BM-MSCs,which became similar to the control group when they were cultured to the third generation.2.The IB MX scheme was the most efficient in chemical induction of BM-MSCs.3.AML-derived BM-MSCs differentiated into neuron-like cells.Both groups of cells were foundthe down-regulated expression of nestin and up-regulated expression of GFAP and tubulin,and there was no significant difference between the two groups.Conclusoin:1.BM-MSCs were cultured and certificated successfully.2.Compared with the control group,the morphology and proliferation of BM-MSCs from AML patients were similar.3.The potential of AML-derived BM-MSCs differentiation into neurons was impaired.