Paroxysmal Nocturnal Hemoglobinuria In Patients With Cd34 + Cd59 On + Cells In Vitro Amplification And Nude Mouse Transplantation Research

Paroxysmal Nocturnal Hemoglobinuria(PNH) is an acquired hemolytic anemia. The main manifestations are recurrent episodes of hemoglobinuria, chronic anemia and thrombosis. The fundamental nature of the disease is the co-existence of abnormal clones with normal clones in patients bone marrow, but the mechanism for this has not been fully elucidated. The current treatment for PNH is far from effective. Except allogeneic bone marrow transplantation, the major clinical efforts are toward to controlling complications rather than interrupting the disease process. This research project is aimed to apply new treatment methods for this disease including autogeneic stem cell transplantation.We generate Hematopoietic Stem Cells (HSC) ex vivo combining with different hematopoietic growth factors and transplant these cells into sublethally irradiated BALB/c mice. In this experiment we investigated the expression and multi-potent differentiation of the HSC. Normal stem (CD34+CD59+) cells were isolated from PNH patients and ex vivo engraftment of CD34+CD59+ cells was performed. We analyzed cell proliferation and hematopoietic reconstitution by transplanting CD34+CD59+ cells into sublethally irradiated BALB/c mice and compared the numbers of CD34+CD59+ cells from patients with PNH and CD34+ cells from normal control in nude mice.CD34+ cells were selected from the bone marrow mononuclear cells of the normal control by immunomagnetic cell sorting. The CD34+ cells were engrafted ex vivo using the best combination hematopoietic factors (SCF+IL-3+IL-6+TPO+EPO+FLT-3). The 7th day was found to be the most suitable time for ex vivo expansion, and the best expansion of CD34+ cells was 57.38 ± 2.50 fold. After ex vivo expansion, CD34+ cells retained strong capability of forming colonies, but their multi-potent differentiation capability was decreased. In the present studies, we transplanted 1 × 10~5 engrafted CD34+ cells into BALB/c mice by i.v or i.p. Mice were killed 6-8 weeks after transplantation, and the bone marrow (BM), spleen, and peripheral blood (PB) were harvested. CD45+ cells from human...