The Role of Cyclophosphamide in the Conditioning Regimen of Stem Cell Transplantation in Iron Overload Mice

Background: Hematopoietic stem cell transplantation(HSCT) is the only effective and reliable method for the treatment of severe thalassemia. Most of the transplant centers adopted Busulfan(Bu)/cyclophosphamide(Cy) pretreatment program, the pediatric Transplantation Center of Southern Medical University used "NF-08-TM" pretreatment program based on Cy/Bu achieved remarkable results.Whether the significant achievements related to cyclophosphamide mobilizing hematopoietic stem cells resulting in an increase efficiency of hematopoietic stem cell clearance, has not yet been reported.

METHODS: Establish three-levels risk grading standards of iron overload with C57 mice by intraperitoneal injection of dextran iron, based on the domestic recognition three-levels risk grading standards of thalassemia major. Eight groups(d1,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. After N-acetylcysteine anti-oxidation, the above indicators were measured.

RESULTS: 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. And the Hepatomegaly Index was increased 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. Similar to the risk criteria of thalassemia classification, three-level risk model of iron overload was successfully established.Compared with normal mice, the peripheral blood of iron overload model mice did not change significantly, while 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.The number of BMNNCs and HPCs in iron overload 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).Compared with normal mice,the cell cycle distribution of HSCs iniron overload model mice did not change significantly, while the proportion of G0 / 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). 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:

The three-level risk model of iron overload was successfully established by treated with different doses of dextran iron intraperitoneally. Iron deposit could lead to long-term hematopoietic stem cell damage, but did not cause significantly changes in peripheral blood.The effect of Cyclophosphamide on mobilization of HSCs in thalassemia transplantation pretreatment regimen may be related to oxidative stress.This study proved initially the advantages of "NF-08-TM" pretreatment in the transplant of thalassemia inour center compared with other transplant centers ,and provided a theoretical basis for further development of individualized pretreatment programs.