The Function Of Cyclophosphamide In The Conditioning Regimen Of Stem Cell Transplantation For ?-thalassemia Major

OBJECTIVE:To explore the role of cyclophosphamide in the conditioning regimen of stem cell transplantation for ?-thalassemia major.METHODS:1.Through the intraperitoneal injection of dextran iron,combined with the domestic recognition three-levels risk grading standards of thalassemia major,establish the efficacy of the model2.Eight groups(dl,d2,d3,d4,d5,d6,d7,d14)were injected intraperitoneally with Cy 50mg/kg for two consecutive days to establish the pretreatment model of transplantation.Peripheral blood cells,BMNNCs,frequencies of different HPCs,HSCs,LT-HSCs in BMMNCs were monitored.The distribution in the cell cycle of HSCs,the level of reactive oxygen species and the microenvironment of HSCs were analyzed by flow cytometry.3.After N-acetylcysteine anti-oxidation,the above indicators were measured.RESULTS:1.Compared with the control group,the Serum ferritin in the low,medium and high dose iron group was increased by 7.6 times,12.8 times and 15.2 times compared with the normal group,Hepatomegaly index by 46.9%,96.5%and 160.2%.Pathological sections showed a large number of iron deposits in the liver,spleen and bone marrow cells,and positively correlated with the dose of iron.Combined with the risk criteria of thalassemia classification,three-level risk model of thalassemia was successfully established.2.Compared with normal mice,the peripheral blood of thalassemia model mice did not change significantly,but the number of WBCs and YM%1d-4d in cyclophosphamide group were significantly decreased(P<0.05),and 7d back to normal;the number of PLT was higher than that of model group and the difference was statistically significant(P<0.05),but there was no significant difference in RBC and HBG.3.The number of BMNNCs and HPCs in thalassemia model mice did not change significantly compared with normal mice,but the number of HSC and LT-HSCs decreased significant(P<0.05);The change rules of BMNNCs and WBC in cyclophosphamide group were the same,and the number of HSCs increased from the first day to the highest in the third day(P<0.05).4.Compared with normal mice,the cell cycle distribution of HSCs in thalassemia model mice did not change significantly,while the proportion of GO/G1 phase cells in cyclophosphamide group was significantly decreased at the 1st day to the third day(P<0.05).But S and G2/M phase change the opposite,the difference was statistically significant(P<0.05).After the treatment with NAC,the decrease of G0/G1 in 1d-3d could be partially reversed(P<0.05).5.Compared with normal mice,the ROS level of the total bone marrow cells,HSCs were significantly higher in moderate-risk model mice and cyclophosphamide group,respectively(P<0.05),and the level of ROS in cyclophosphamide groups increased from the first day,highest in the second day,and then back to normol.After NAC treatment,the level of ROS in cells decreased compare with before respectively(P<0.05).CONCLUSION:1.By intraperitoneal administration different doses of dextran iron,three-level risk model of thalassemia was successfully established.It could damage hepatic,splenic and bone marrow hematopoietic function,but did not cause significantly changes in peripheral blood.2.cyclophosphamide 50mg/kg for two consecutive days in the hematopoietic stem cell transplantation treatment of thalassemia pretreatment program,not only cause transient,reversible myelosuppression,but also lead to more resting state of hematopoietic stem cells into the cell cycle to proliferate,playing a role in mobilization of hematopoietic stem cells,and the largest mobilization in d4.The results provide molecular level of theoretical basis for the best time to convergence of cyclophosphamide/busulfan in the pretreatment program of the clinical study in transplantation.3.The effect of cyclophosphamide on mobilization of HSCs in thalassemia transplantation pretreatment regimen is related to oxidative stress.4.The study proved initially the advantages of "NF-08-TM" pretreatment in the transplant of thalassemia in this center compared with other transplant centers,and provided a theoretical basis for further development of individualized pretreatment programs.