| OBJECTIVE:To explore the feasibility of bone marrow transplantation after mobilization of bone marrow hematopoietic stem cells in transplant recipient mice pretreated with granulocyte colony stimulating factor(G-CSF),and to establish a bone marrow transplantation method based on G-CSF pretreatment.To observe whether this method can achieve long-term stable transplantation effect and whether it can be applied to platelet-targeted gene therapy.To explore whether other mobilizers other than G-CSF can construct a successful bone marrow transplantation model based on hematopoietic stem cell mobilization.Method:Through controlled experiments,the effects of G-CSF treatment time and doses on mobilization of bone marrow hematopoietic stem cells were observed,and a method for G-CSF pretreatment to mobilize bone marrow hematopoietic stem cells was established.Using 2bF9-R338L-HB transgenic mice as donor mice and Hemophilia B mice as recipient mice,bone marrow transplantation experiments after G-CSF pretreatment were performed,and the efficacy of the transplantation was evaluated.After pretreatment with G-CSF,the recipient mice were injected with different numbers of 2bF9-R338L-HB mouse bone marrow mononulcear cells.Three recipient mice groups were established by injecting 1×10~8cells,1×10~7cells and 1×10~6cells into each mouse.The egraftment of the donor cells in the recipient mice was evaluated.Genomic DNA of blood was extracted,and 2bF9-R338L gene specific PCR was performed to identify if there were donor cell-derived blood cells in the recipients.Quantitative PCR was performed to determine the copy number of 2bF9-R338L gene in transplant recipient mice.The antigen level of coagulation factor IX(FIX)in mouse platelets was detected by FIX ELISA,and the activity of platelet FIX was determined by a FIX chromogenic assay.Confocal laser immune-microscopy was used to determine the localization of coagulation FIX in platelets.Flow cytometry was used to observe the ratio of FIX positive platelets in the total platelets.The expression of FIX in megakaryocytes in the bone marrow of recipient mice was determined by immunohistochemical analysis.Secondary bone marrow transplantation was performed to investigate whether the long-term hematopoietic stem cells of donor were successfully engrafted in the recipient mice.Transplantation efficiency of different groups was compared and the influence of transplanted cell number on donor cell engraftment was accessed.Finally,the effect of hematopoietic stem cell mobilizers AMD3100 and CASIN on bone marrow transplantation was evaluated.Results:2bF9-R338L gene was detected in the blood genomic DNA of all three transplantation groups,and the expression level of FIX protein in platelets decreased with the decrease of the number of transplanted cells.The expression levels of FIX in the primary recipient mice of 10~8cell group ranged from 0.42±0.15 mU/10~8plts to0.97±0.41 mU/10~8plts during 20 weeks after transplantation.The FIX activities were4.14±2.31 mU/10~8 plts and 10.79±6.49 mU/10~8 plts in week 2 and week 12,respectively.the corresponding activity to antigen ratio were 12.04±1.83 and 11.90±4.40,respectively.FIX protein was observed in megakaryocytes in the femur and platelets of the recipient mice.The average copy number of 2bF9-R338L gene in perip Heral blood DNA of recipient mice was 0.11±0.11copies/cell.The average proportion of FIX positive platelets in all platelets was 20.28±18.21%.The platelet FIX level of secondary transplantation recipient of 10~8 cell group was between 0.34±0.03 mU/10~8plts and 0.47±0.1 mU/10~8plts during 8-16 weeks after transplantation.The FIX activities were 5.83±1.28 mU/10~8 plts and 4.65±1.42 mU/10~8 plts at week12 and 16,respectively,and the corresponding activity to antigen ratios were 13.13±3.36 and 7.53±0.84,respectively.The stable expression of FIX protein was also detected in the platelets of 10~7cell transplantation group,and a very low expression of FIX was detected only in week 4 of 10~6 cell transplantation mice.2bF9-R338L gene was detected in the perip Heral blood DNA of transplantation recipients pretreated by CASIN,but no FIX was detected in the mouse platelets.2bF9-R338L gene was not detected in the mice pretreated by G-CSF combined with AMD3100 and no FIX was observed in the platelets of the recipients.Conclusion:1.The optimal dose of G-CSF is 0.25μg/g mouse body weight per day for four consecutive days;2.Bone marrow transplantation after G-CSF preconditioning can achieve long-term stable engraftment of donor hematopoietic stem cells,but compared with traditional preconditioning methods,a larger number of donor cells needs to be transplanted;3.FIX can be found in the bone marrow megakaryocytes and platelets of transplantation recipient mouse;4.The preliminary results showed that the effect of AMD3100 and CASIN as pretreatment drugs was not as good as that of G-CSF. |
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