The appearance of apoptotic cells in spleen of PK-1slc mice was examined by terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL) staining. We also assayed hematopoietic stem cell colony formation in vitro in the spleen of PK-1slc mice, to investigate erythropoiesis, and annexin V binding, as a measure of apoptotic cells in constitutive erythroid colonies, to evaluate the maturation of erythroid progenitors.
The number of hematopoietic progenitors including colony-forming unit erythroids, burst-forming unit erythroids (BFU-E), colony-forming unit granulocyte-macrophages, and multilineage colony-forming units in the spleens of PK-1slc was remarkably increased indicating hematopoiesis, and enhanced erythropoiesis in particular. TUNEL assays identified apoptotic cells in the splenic red pulp of the PK-1slc mice. Two-color flow cytometry detected apoptotic cells among anti-TER119-positive cells, suggesting that apoptotic cells were of erythroid lineage. Cells undergoing apoptosis were detected in cultures of BFU-E generated from bone marrow cells of PK-1slc mice.
The results in this study indicate that the metabolic disturbance in PK deficiency alters not only the survival of red cells but also the maturation of erythroid progenitors, resulting in ineffective erythropoiesis.
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