Extensive Apoptosis And Ineffective Hematopoiesis In Megaloblastic Anemia And Myelodysplastic Syndromes

Objective To investigate the proliferation and apoptosis of megaloblastic cells and myelodysplastic hematopoietic cells, and elucidate the role of massive apoptosis in bone marrow ineffective hematopoiesis of megaloblastic anemia and the myelodysplastic syndromes (MDS) thoroughly.Material and methods(1) Bone marrow CD34 positive cells were enriched from 15 MDS patients and 10 megaloblastic anemia patients using immunomagnetic beads (MACS), and apoptosis was measured by cellular morphology, flowcytometric-DNA analysis and in situ end labeling (TUNEL) respectively. (2) Bone marrow CD₃₄⁺ cells from megaloblastic anemia patients were cultured in folate-deficient medium, different doses of folic acid and thymidine(TdR) were added , proliferation and apoptosis of megaloblastic cells were detected on 1st, 7th, 10th, 14th day of culture. (3) Apoptosis of MDS marrow CD₃₄⁺ cells and the expression of bcl-2, c-myc, apo-1/fas gene products in vitro were detected on 1st, 7th, 10th, 14th day of culture , and the effects of different doses of rhEpo, rhG-CSF, rhGM-CSF, rhIL-3 on them were also observed.Results(1) Extensive apoptosis of megaloblastic anemia hematopoietic progenitors was indentified in the course of proliferation and differentiation, the percentages of apoptosis was 8.71±1.41%, 15.02±3.75%, 28.99±7.22% on 7th, 10th, 14th cultured day respectively in megaloblastic anemia patients, and 5.80±2.62%, 9.12±3.45%, 15.31±6.81% respectively in control group, the difference between the two groups was remarkable(p<0.05). (2) On 10th day , megaloblastic CD₃₄⁺ cells in folate-deficiency medium exhibited an increased duration of the S-phase of cell cycle, on 14th day, the cells exhibited a substantially growth arrest and followed by massive cell death. (3)Addition of folic acid to the folate-deficient medium prevented the apoptosis and permitted proliferation and differentiation of megaloblastic progenitors, a dose-dependent effect was found. Too much thymidine as well as too little thymidine in vitro led to megaloblastic cell apoptosis, 10μM thymidine was best in preventing apoptosis and permitting proliferation.(4) Extensive apoptosis of MDS hematopoietic progenitors was also indentified in the course of proliferation and differentiation, the percentages of apoptosis was 20.89±4.13%, 34.85±2.60%, 50.44±7.17% on 7th, 10th, 14th cultured day respectively in MDS patients, and 5.58±2.82%, 12.38±2.40%, 18.93±4.72% respectively in control group, the difference between the two groups was remarkable (p<0.05). (5) 26.32±10.25% of fresh marrow CD₃₄⁺ cells from MDS patients expressed...