| Objective Hematopoietic stem cell transplantation is one of the most effective method for the treatment of leukemia, lymphoma, malignant blood tumor, metabolic diseases, autoimmune diseases and other diseases hazards to human health. The premise of peripheral blood hematopoietic stem cell transplantation therapy is acquisition of adequate hematopoietic stem/ progenitor cells which are mobilization from the bone marrow into peripheral blood. At present various cytokines such as granulocyte colony stimulating factor, chemotherapy reagent and chemotactic factor have been widely used for HSPC mobilization, but the number of HSC still can not meet the clinical treatment, and HPSC mobilization mechanism has not been fully elucidated. It is necessary to deeper study on the mechanism of HSPC mobilization, for production more effective mobilization reagent, and for deeper understanding of molecular mechanism of HSPC mobilization.Methods In this study, we used recombinant adenovirus which carry Ang1 gene. And the C57BL/6 mice were used as the research model. The recombinant adenovirus was injected to the mouse through tail vein. The mouse peripheral blood was detected by ELISA, flow cytometry, blood cell count, hematopoietic progenitor cell culture after injection of recombinant Angl adenovirus or blank vector. The peripheral blood serum and the BM concentration of Ang1, the proportion of LSK cell, the WBC and the CFU were observed after injection with adenovirus Angl or blank vector. The effect of plasma elevation of Ang1 on HSPC mobilization was investigated.Results1. The titer of the recombinant adenovirus was 1010 pfu/ml; After transfected 293T cells by recombinant adenovirus, the GFP gene express successly, and the supernatant contained Angl protein.2. After injection of recombinant adenovirus Angl, concentration of mice peripheral blood serum of Ang1 was gradually increased. At d14 it was increased fastestly. The serum of Angl from d14 to d42 were significantly higher than d0 (P<0.05); But after injection of recombinant adenovirus Angl, the mouse bone marrow of Angl concentration change was not obviously. The concentration of Angl from dl to d28 was wander at baseline up and down. But it was increased at d35 and d42 compared to the baseline. It was increased significantly at d42 compared to d0 (P<0.05)3. After injection of Angl combinant adenovirus or empty adenovirus, the WBC was increased, take the PSC group (d0) mice peripheral blood WBC as the baseline values (5.38±2.32) ×109/L. After injection of Angl combinant adenovirus into mice the peripheral blood WBC was increased, and the WBC of dl4 was the highest point (13.07±1.26) ×109/L (P=0.02), and the WBC from d7 to d42 were higher than those in d0 (P<0.05), but the group d42 is still not approaching the baseline; After injection of negative control virus, mice peripheral blood WBC increased, it was reached the peak value at d28 (13.52±3) x109/L (P=0.021), also the WBC from d7 to d35 were higher than those in d0 (P<0.05), even the d42 was increased but there was no difference with d0(P=0.698).4. After injection of different recombinant adenovirus into mouse,the mobilization effect was investigated. LSK cells in nucleated cell ratio was detected by flow cytometry.The injection of PBS group (d0) was taken as the baseline value as (0.056±0.013)%. When injection of recombinant adenovirus Angl group, the LSK cell ratio highest value was d14, and LSK cell ratio from d7 to d21 were increased compare with d0 group (P< 0.05); When injection empty adenovirus, the LSK cell ratio of d10 reached the highest value (0.107±0.026)%, and the LSK cell ratio from d10 to d21 were increased compare with d0 group (P<0.05). Comparison injection the Angl recombinant adenovirus with the blank adenovirus at the same time-point, the LSK cell ratio at d10, d14, d21 of Ab-Angl was higher than that of Ab-CTL (P<0.05).5. Taken d14 time-point for hematopoietic progenitor cells culture assay. The injection of Angl recombinant adenovirus group of colony forming units (CFU) was (8.16±4.62/1×105MNC)compared with PBS group (3.0±3.35/1×105MNC), there was statistically difference (P= 0.002). And the Ab-CTL group was (4.0±2.82/1×105MNC), the difference was significant compare to Ab-Ang1 group (P= 0.009). Eventhough the Ab-CTL group was higher than those in the PBS group, but there haven no difference (P=0.511).Conclusion After injection Angl recombinant adenovirus, the serum concentration of Angl was increased, which lead to mobilize hematopoietic stem/progenitor cells. |
