| Objective: This study used cell culture,FCM,RT-PCR,Westen Blot,ELISA for detection of quantity and function of osteoblasts in Myelodysplastic syndrome(MDS)patients.And the signaling pathways revealed abnormal changes of osteoblasts in MDS,further more explored the osteoblasts in the MDS role in the pathogenesis of hematopoietic function.In this study,we investigated the quantity and function of osteoblasts by culture in vitro,detected of changes in signaling pathways,and the relationship between progression in MDS patients.To prove osteoblasts play an important role in bone marrow niche in MDS.Background: MDS represents clonal disorders mainly of the elderly which is characterized by ineffective hematopoiesis and an increased risk of transformation into acute myeloid leukemia.Recent studies indicated that changes of bone marrow niches,such as abnormal interaction with hemopoietic stem cell(HSC)or malignant clone,abnormal secretion of hematopoietic growth factor and cytokines,contributed to the pathogenesis of MDS.It was found that mesenchymal cells in the bone marrow niche,including osteoblasts and osteoprogenitor cells,reticular cells and fat cells,play an important role in hematopoiesis regulation.The osteoblastic cells are critical for early differentiation of stem cells.The demise of osteoblasts,can accelerate the progress of some malignant hematological diseases.As an important part of the bone marrow niche,osteoblasts play an important role in the progression of MDS.Materials and Methods: Thirty-eight untreated patients with MDS in the Hematology Department of General Hospital,Tianjin Medical University were enrolled in this study.They were diagnosed according to WHO classification.They were divided in two groups according to WPSS score.There were 18 patients in Group 1: WPSS score 0~2,and 20 patients in Group 2: WPSS score 3~6.Twenty-five healthy donors were enrolled as controls.In this study,we investigated the quantity and function of osteoblasts by culture in vitro,detected of changes in signaling pathways with Flow cytometry(FCM)and RT-PCR,and the relationship between progression in MDS.ELISA detection of MDS patients bone marrow supernatant and osteoblast culture medium from MDS patients expression of TGFbeta,the detection of ?-catenin/ phosphorylation ?-catenin with Western blot blot.To explore the role of osteoblasts in bone marrow niche in MDS patients.Results:1.Bone marrow culture method and conditioned medium were used to culture and purify osteoblasts in the bone marrow of MDS patients.In MDS patients(6.29±6.57% vs13.01±6.29%,p<0.05),especially in high-risk MDS(5.39±5.96% vs 13.01±6.29%,p<0.01),the number of progenitor cells in the bone marrow was lower than that in the normal control group.The number of osteoblasts(4.62±0.62 vs 6.79±0.79,p<0.05)decreased in the high risk MDS.Patients with MDS(3.61±0.47% vs 6.55±1.46%,p<0.05)especially in high-risk MDS(2.83±0.44% vs 6.55±1.46%,p<0.05)MDS osteoblasts from patients with OCN~+CD34~-LIN~-ratio were significantly lower than the normal group and the expression of bone morphogenetic protein 2(BMP2)compared with the normal control group(2.51±0.51 vs 4.37±1.14,p<0.05)was significantly decreased.The proportion of normal osteoblasts in MDS patients was inversely proportional to the initial cells in the bone marrow,which was proportional to the absolute value of the neutrophils and the hemoglobin concentration.2.Normal bone marrow cells were co cultured with MDS patients and normal bone cells,stem progenitor cells,MDS stem and progenitor cell colony formation less than the normal group,the formation of CFU-GM is lower than normal group(6.5±3.57 vs 17.9±7.11,p<0.01).MDS group of bone marrow cells HES1(3.90±1.23 vs 1.72±0.49,p<0.05)and CyclinD1(20.24±5.00 vs 6.80±2.85,p<0.05)increased expression of the tumor associated genes.Separation of normal CD34~+ cells were co cultured with MDS patients and normal bone cells,detect the apoptosis of MDS group was higher than that of the normal group(19.51±5.89% vs 5.84±4.15%,p<0.001),while the proportion of LSC in MDS group was significantly increased(6.80 ±0.89% vs 2.50±1.56%,p<0.001)3.In osteoblasts cultured from MDS patients,Jagged1 showed(24.34±13.27% vs 12.54±5.89%,p<0.05),RANKL showed(3.68±2.60 vs 2.24±2.09,p<0.05)and HES1 showed(3.23±0.78 vs 1.71±0.44,p<0.05),CyclinD1 showed(6.17±1.34 vs 2.02±0.73,p<0.05)expression.In the culture system with Notch pathway inhibitor DAPT,HES1 gene expression was significantly decreased(3.39±0.85 vs 0.38±0.15,p<0.01),and the target gene CyclinD1 Wnt pathway increased the expression of(9.15 ±2.27 vs 48.40±6.82,p<0.01).MDS patients with bone marrow supernatant(268.98±113.19 vs 366.96±218.64,p<0.05)and osteoblasts cultured in(50.49±27.49 vs 80.54±29.11,p<0.05)decreased,TGF-? content increased after treatment with beta,beta TGF-? content in bone marrow of patients with CR(600.21±404.95 vs 366.96±218.64,p<0.05)in bone marrow supernatant,even more than the normal control the level of group.The expression of ?-Catenin in osteoblast of MDS patients was increased,especially phosphorylated ?-Catenin for it was the active form.Conclusion:1.Bone marrow culture method was used to culture and purify osteoblasts in MDS patients.The number of progenitor cells in bone marrow of patients with MDS was lower than that in normal control group.The number of osteoblasts in MDS patients was decreased and the osteogenic ability was significantly lower than that of normal control group.2.Osteoblasts in MDS patients,increased the expression of HES1 and CyclinD1 in bone marrow cells,inhibited the proliferation and differentiation of normal bone marrow stem cells,and induced the production of malignant clonal cells.3.In the bone marrow and osteoblast culture medium of MDS,the content of TGF-? was decreased,the expression of RANKL,Jagged1 and HES1,CyclinD1 were increased,the expression of ?-Catenin was increased,and the related signal pathway was activated.4.The abnormal changes of the quantity,function and signal pathway of MDS patients may change the bone marrow niche in patients with MDS. |
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