延迟连续骨髓移植对主要H-2不合小鼠移植后急性移植物抗宿主病的影响

Effect of delayed sequential bone marrow transplantation on acute graft-versus-host disease in major H-2 incompatible mouse transplantation

目的研究延迟连续骨髓移植对主要H-2不合小鼠移植后急性移植物抗宿主病（aGVHD）的影响。方法以近交系C57BL／6（H-2^b）小鼠为供鼠、BALB／c（H-2^d）小鼠为受鼠，受鼠接受总剂量为8．0Gy ^60Coγ射线全身照射（TBI）后，实验分为5组：①空白对照组（只输注0．4ml RPMI1640）；②经典移植组（TBI后4h输注）；③连续移植组（TBI后4h，1、2、3d连续输注）；④延迟移植组（TBI后4d输注）；⑤延迟连续移植组（TBI后4、5、6、7d连续输注）。移植后用流式细胞术检测各组受鼠H-2^b细胞植入水平，ELISA法测定IL-2、IL4、IL-6、IL-10、IFN-叮的血浆浓度，比较各组的生存率、aGVHD发生情况及造血重建情况。结果经典移植组出现典型aGVHD表现，均于3周内死亡，连续移植组、延迟移植组60d生存率分别为30％、50％，延迟连续移植组出现aGVHD的小鼠数量少、程度轻，60d存活率70％，高于其他组（P〈0．05）。延迟移植组和延迟连续移植组血浆IL-2、IFN-γ、IL-4、IL-10分泌高峰较经典移植组延迟5～10d，IL4、IL-10的表达高于经典移植组（P〈0．05），IL-2、IFN-γ的表达低于经典移植组（P〈0．05）。各组血浆IL-6浓度均于TBI后5～10d达高峰，且经典移植组的表达高于其他组（P〈0．05）。延迟连续移植组60d时H-2^b细胞的百分率为（98．1±1．1）％，20d时外周血白细胞计数恢复正常。结论延迟连续骨髓移植可减轻小鼠异基因移植后的aGVHD，提高生存率，不影响骨髓植入和造血重建，是合适的移植方式，其减轻aGVHD机制可能与减少T细胞Ⅰ类细胞因子分泌，促进Ⅱ类细胞因子分泌有关。

Objective To study the effect of delayed sequential bone marrow transplantation on acute graft-versus-host disease （aGVHD） in major H-2 incompatible mouse transplantation. Methods C57BL/6 （ H-2b ） mice were used as donors and BALB/c（ H-2^d） mice as recipients. BALB/c mice were given 8.0 Gys total body irradiation （TBI） on day 0 and infused with a blend of bone marrow cells and spleen cells in different time. Transplantation was carried out as follows： group Ⅰ TBI on day 0 and transplantation at 4 h after TBI;groups of Ⅱ TBI on day 0 and transplantation at 4 h, dl ,d2,d3 after TBI;groups Iil TBI on day 0 and transplantation at day 4 after TBI;groups Ⅳ TBI on day 0 and transplantation at day 4 through day 7 after TBI. Recipient＇ s spleen H-2b cells were detected by flow cytometry and the level of serum cytokines （ IL-2, IL-4,IL-6,IL-10 and IFN-γ） by ELISA. The survival, aGVHD and hematopoietic recovery were observed. Results aGVHD occurred in group Ⅰ and the mice all died within 3 weeks after transplantation. The 60 day survival rates of groups of Ⅱ and Ⅲ were 30% and 50% respectively. The degree of aGVHD in group Ⅲwas modest and the survival rate was higher than that in other groups （ P 〈 0.05 ）. The peak time of IL-2 ,IFN-γ, IL-4 and IL-10 in groups Iil and IV were later than that in group Ⅰ, The levels of IL-4 and IL-10 in groups Iil and IV were higher than that in group Ⅰ and for the levels of IL-2 and IFN-γ were on the contrary（ P 〈 0.05）. The level of IL-6 in all groups peaked on day 5 to day 10 after TBI and was higher in group I than in others （ P 〈 0.05）. In group IV the mean value of donor H-2b cells was （98.1 ± 1.1 ） % on day 60 and WBC counts recovered normal on day 20. Conclusions Delayed sequential transplantation can reduce the morbidity of aGVHD ,improve the survival rate and not affect the engraftment and reconstitution of hematopoi esis in mouse allo-BMT. The mechanism of aGVHD prevention may be related to the reducing of type 1 cytokines of T lymphocyte and the increasing of type 2 cytokines.