| Objective:Through the measurement of the proportion of bone marrow macrophage accounted for bone marrow mononuclear cells,the ratio of macrophages type 1 and type 2,the expression of type 1 and type 2 macrophage surface antigen in the healthy control group and different risk groups of patients with Myelodysplastic Syndrome,and by in vitro stimulation of M1 macrophages,and detection of IL-1 beta and TNFalpha m RNA expression,explore the effect of macrophage in the pathogenesis of Myelodysplastic Syndrome.Background:MDS is a group of heterogeneous myeloid clonal diseases originating from hematopoietic stem cells.The main features of the disease are the reduction of whole blood cells and the high risk evolving to acute myeloid leukemia.The advanced age of MDS patients(the average age at 70-75 years old)makes the management more complex,even with non hematological complications in elderly patients,and the tolerance for some drastic treatment is always poor.In addition,for patients evolving to AML,the efficacy of standard treatment was lower than that of patients with AML.Although in recent years,the treatment of MDS has made some progress,lenalidomide,azacitidine,decitabine and hematopoietic stem cell transplantation therapy prolongs the survival of patients,but the situation of MDS patients with poor prognosis,short survival and lack of effective treatment have not fundamentally changed.Anemia,bleeding,infection and other complications significantly affect the quality of life of patients,and can directly lead to the death of the patient.Therefore,the researchers at home and abroad carry on a lot of experiments to expore the pathogenesis of Myelodysplastic Syndrome,they found that age-induced genetic,epigenetic,and immune-mediated changes in haemopoietic stem cells(HSC)lead to oligoclonal expansions of myelodysplastic stem cells,with defective differentiation,characterised by increased apoptosis of erythroid and myeloid progenitors.Microenvironmental changes and immune deregulation contribute to this differentiation defect.In recent years,tumor microenvironment is one of the hot spots in tumor pathogenesis and treatment.The tumor microenvironment is composed of cells,signal molecules,extracellular matrix, cytokines and so on.At present,domestic and foreign scholars believe that the tumor cells alone do not result in disease,but through the "recruitment" of normal cells,secretion of inhibitory cytokines and inhibiting the immune cell killing,to form microenvironment that is benefit to the growth and metastasis of tumor cells.In recent years,it has been found that macrophages are also the components of hematopoietic microenvironment,which affect the migration and settlement of hematopoietic stem cells.In addition,the abnormal accumulations of monocytes were found in MDS,which affected the migration of hematopoietic stem cells.Therefore the abnormalities of MDS bone marrow macrophages may play an important role in the pathogenesis of MDS.After the polarization to M2,the macrophages can inhibit the tumor immunity through a variety of ways.The researchers found that the M2 macrophages are tumor associated macrophages(TAMs).On the other hand,the study found that TAMs can inhibit the killing function of NK cells and CTL cells.The expression of HLA-C,HLA-E,HLA-G and other membrane binding molecules of TAMs can inhibit the activation of NK cells.Macrophages express the ligands of the inhibitory receptors programmed cell death protein 1(PD-1)and cytotoxic T-lymphocyte antigen 4(CTLA-4).These inhibitory ligands are normally upregulated in activated immune effector cells such as T cells,B cells,and NK T cells as part of a safety mechanism that controls the intensity of the immune response and as part of inflammation resolution.Activation of PD-1 and CTLA-4 by their ligands can surpress the cytotoxic effect of T cells.Materials and MethodsFirst,we use flow cytometry(Flow Cytometry,FCM)to test 38 MDS patients(LR-MDS 18,HR-MDS 23)and 21 normal control CD14+ monocytes of the bone marrow and CD14 the average fluorescence intensity(MFI),and then mark macrophage with CD14 and CD68,M1 macrophages with CD64 and CD40,M2 macrophages with CD206 and CD163.We compare the difference of CD14+ monocytes,M1/M2 and the expression of CD206,CD163,CD40,CD64 in macrophages between MDS patients and the normal control.Then,we extracted the m RNA of two groups of M1 macrophages by Ficoll method,and compared the expression of IL-1β and TNF-α between the two groups.By comparing the difference of macrophages between two groups in bone marrow microenvironment,we lay the foundation for exploring the role of macrophages in the pathogenesis of MDS.Result:1.The difference of monocytes between MDS group and normal control group1.1 The proportion of monocytes in bone marrowThe proportion of bone marrow monocytes in normal control group was(2.11 + 0.93)%,the proportion of bone marrow monocytes in LR-MDS group was(1.96±1.53)%,in HR-MDS group was(3.66±3.38)%.There was no significant difference in the proportion of bone marrow monocytes cells between the normal control group and the low risk group,and the proportion of bone marrow mononuclear cells was significantly higher in the high-risk group compared with the control group(P < 0.05).The proportion of high-risk group was lower than that of the low-risk group,the difference was statistically significant(P < 0.05).1.2 The mean fluorescence intensity of CD14The CD14 MFI of bone marrow monocytes in normal control group was 639.05 ±359.78,the CD14 MFI of bone marrow monocytes in LR-MDS group was 501.43±374.44,in HR-MDS group was 458.26±306.72.There was no significant difference in the CD14 MFI of bone marrow monocytes cells between the normal control group and the low risk group,and the CD14 MFI of bone marrow mononuclear cells was significantly lower in the high-risk group compared with the control group(P < 0.05).The difference between low risk group and high risk group has no statistically significant.2 The difference of macrophages between MDS group and normal control group2.1 The proportion of M1 and M2 macrophages accounted for the bone marrow monocytesThe proportion of M1 macrophages accounted for the bone marrow monocyte in the normal control was(6.41±7.09)%,in the low risk MDS group was(8.08±10.31)%,and in the high risk MDS group was(7.80±9.41)%.There are no statistically difference among three groups.The proportion of M2 macrophages accounted for the bone marrow monocyte in the normal control was(1.82±2.47)%,in the low risk MDS group was(3.18±3.79)%,and in the high risk MDS group was(3.93±3.81)%.The proportion of that in high risk group was significant higher than that in normal group,the difference was statistically significant(P < 0.05).2.2 The ratio of M1 and M2 of bone marrow macrophagesThe ratio of M1 macrophages and M2 macrophages in the normal group was 3.50±3.22,in the low risk group was 1.68±0.78,and in the high risk group was 1.80±0.88.The ratio of M1 macrophages and M2 macrophages in the normal group was significant lower than that in the low risk and high risk MDS groups(P < 0.05).There are no significant difference in the ratio of M1 macrophages and M2 macrophages between high risk group and low risk group.2.3 OthersThe expressions of CD64,CD40,CD206 and CD163 on the surface of bone marrow macrophages in MDS were not statistically different from those in normal control group.3 The differences in expression of IL-1 beta and TNF-alpha mRNA of M1 macrophages between MDS group and normal control group3.1 Expressions of IL-1 beta m RNA of M1 macrophages in MDS group and in normal control groupExpressions of IL-1 beta mRNA was 2.07±1.66 in normal control group,0.5±0.6 in the low risk group,and 0.98±0.72 in the high risk group,compared with the low risk group and the high risk group,the expressions of IL-1 beta m RNA were higher,and the difference was statistically significant(P < 0.05),while the difference between the low and the high risk group has no statistical significance.3.2 Expressions of TNF-alpha mRNA of M1 macrophages in MDS group and in normal control groupExpressions of TNF-alpha mRNA was 1.20±0.75 in normal control group,0.55±0.62 in MDS group,and 0.98±0.72 in the high risk group,compared with the MDS group,the expressions of IL-1 beta m RNA in normal group were higher,and the difference was statistically significant(P < 0.05).Conclusion:(1)The accumulation of abnormal monocytes in MDS patients with BM may be related to the progression of the disease;(2)Compared with the normal control group,the proportion of M2 macrophages in monocytes was significantly higher in MDS patients,and the proportion of M2 in bone marrow mononuclear cells in patients with MDS was further increased with the development of disease;(3)The bone marrow M1/M2 of patients with MDS had significant difference compared with the normal control group,suggesting that MDS patients had abnormal macrophage polarization;(4)The abnormality of bone marrow macrophage polarization in MDS patients may be related to the occurrence and progression of MDS disease,but the specific mechanism remains to be further studied. |
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