The Stimulative Effect Of RhBMP-2m On The Bone Marrow Hematopoiesis Of Mouse With Irradiation Injury

Bone marrow hematopoietic cell was sensitive to the 7 radial which could induce cell apoptosis and make the number of hematopoietic stem cells decreased. It was demonstrated that the number of hematopoietic stem cell was the pivotal factor in the process of hematopoietic reconstitution. On the surface of hematopoietic stem cell, CD34 antigen was the specific marker which represents the status of bone marrow hematopoiesis. Additionally, it was found that there were BMP I and II receptors on the surface of hematopoietic stem cell. Bone morphogenetic protein which was a factor of hematopoietic regulation could induce efficient hematopoietic differentiation of embryonic stem cells. If BMP gene was knocked out, hematopoiesis of embryo would be blocked. In our previous experiments, we have found that rhBMP-2m could increase the survival rate of mouse with irradiation injury, however the mechanism was still not clear. Therefore, our experiments were designed to observe the stimulative effect of rhBMP-2m on the bone marrow hematopoiesis of mice with irradiation injury and explore the possible mechanism.1. The animal model of bone marrow hematopoietic injury was established through irradiating the mice with 60Co 7 radial at the total dose of 6Gy and the dose rate of 1.45Gy/min. Ficoll-Hypaque density gradient centrifugation was used to prepare the cell suspension of bone marrow and the color of cell suspension was observed. The number of mononuclear cells was counted and the protein concentration wasmeasured, then analysising the difference among three groups. The results showed that the number and the protein concentration of mononuclear cells decreased significantly after irradiation (p<0.01) as compared with the normal group. The color of cell suspension also has obvious variety. After treating with rhBMP-2m, the number and the protein concentration of mononuclear cells were much more than those of irradiation group (p<0.05) .2. Through irradiating the mice with 60Co y radial and treating with rhBMP-2m, the expression of CD34 antigen on the surface of bone marrow mononuclear was observed by the method of ELISA; Flow cytometry was used to observe the percentage of CD34+ cells and analyse the difference between control and treated groups. The results showed that the expression of CD34 antigen and the percentage of CD34+ cells decreased significantly after irradiation (p<0.01) as compared with the normal group; After treating with rhBMP-2m, they were higher than those of irradiation group (p<0.05) .3. Through irradiating the mice with 60Co y radial and treating with rhBMP-2m, acridine orange staining and flow cytometer based on PI staining were used to observe the cell apoptosis. Fluo-3-based laser confocal scanning microscopy was used to measure the changes in intracellular calcium content of bone marrow mononuclear cells and analyse the difference between control and treated groups. The results showed that the number of apoptotic cells and the intracellular calcium content increased significantly after irradiation (p<0.01) as compared with the normal group; After therapy with rhBMP-2m, they were lower than those of irradiation group (p<0.05) .The results of our experiments showed that 60Co y radial could injure the hematopoietic function of adult animal bone marrow, it was possible that BMP could be recognized by the BMP I and II receptors on the surface of bone marrow hematopoietic cells, by which the phosphorylational effect and the process of signal transmitting were induced in order to reduce the transport of calcium and intracellularcalcium content and restrain the apoptosis effects induced by irradiation, at the same time, regulate the expression of CD34 antigen on the surface of bone marrow mononuclear and increase the number of CD34+ cells in bone marrow. Through modifying the status of bone marrow hematopoiesis and stimulating the proliferation of hematopoietic stem cell, the recovery of bone marrow hematopoietic injury was accelerated and the period of hematopooetic reconstirution was reduced.