致敏受体排斥异基因供体骨髓细胞的实验研究

Experimental study on rejection of allogeneic donor bone marrow cells in sensitized recipients

目的建立致敏动物模型，研究致敏对异基因骨髓细胞植入的影响及机制。方法将C57BL／6小鼠脾细胞经尾静脉注射到BALB／c小鼠体内建立致敏动物模型，应用二抗结合实验及补体依赖细胞毒性反应检测血清抗体。以非致敏或致敏BALB／c小鼠为受鼠，经照射预处理后予以1×10^7C57BL／6供鼠骨髓细胞。移植后于不同时间点（2、12和48h）分离受鼠外周血、脾脏及骨髓等细胞，检测供鼠细胞在致敏受鼠体内各组织的分布。移植后记录各组受鼠的生存情况，监测造血重建与骨髓恢复情况。体外分离非致敏或致敏受鼠的血清及脾细胞，与异基因骨髓细胞相孵育，通过免疫实验计算细胞毒性指数。结果二抗结合实验和补体依赖细胞毒性反应均证实致敏受鼠血清中含有高滴度的供鼠反应性抗体。骨髓移植归巢实验结果表明，与非致敏组相比，异基因骨髓细胞在致敏受鼠的外周血、脾脏及股骨的分布均明显减少。生存分析结果发现非致敏组小鼠于照射后能长期存活，而致敏组小鼠于照射后12～15d全部死亡。移植后第14天，非致敏组受鼠外周血白细胞和股骨骨髓细胞计数分别为（3240±300）×10^6/L和（396±27）×10^6／股骨，而致敏组受鼠外周血白细胞和股骨骨髓细胞计数分别为（320±80）×10^6/L和（6±2）×10^6／股骨，两组差异有统计学意义（P〈0．01）。移植后第7天，供鼠细胞在非致敏组和致敏组骨髓所占的百分比分别为（48．07±4．70）％和（0．77±0．11）％，两者差异有统计学意义（P〈0．01）。体外通过补体依赖细胞毒性反应实验、细胞毒性淋巴细胞的杀伤作用和抗体依赖细胞介导细胞毒性作用实验表明，敛敏组受鼠的细胞毒性指数均明显高于非致敏组。结论成功建立脾细胞输注致敏的小鼠动物模型，致敏受体完全排斥异基因供体骨髓细胞，作用机制与免疫损伤途径有关。

Objective To establish a murine model of sensitization, and investigate the effeet and mechanism of sensitization on allogeneic donor bone marrow cells （BMCs）. Methods Sensitized BALB/c mice were established by transfusions of allogeneic splenocytes. The donor reactive antibodies were detected by binding and complement-dependent cytotoxicity assays. After irradiation, 1 × 10^7 BMCs of C57BL/6 donor mice were injected into non-sensitized or sensitized BALB/c recipient mice. The distribution pattern of donor BMCs in peripheral blood, spleen and bone marrow of recipient mice were analyzed at different time points （2 h, 12 h and 48 h） post transplantation. Hematopoietic recovery post transplantation was assessed, and survival was monitored. Moreover, sera and splenocytes derived from non-sensitized or sensitized recipients were incubated with allogeneic BMCs in vitro, and the cytotoxic indexes were calculated in the immune experiments. Results The binding and complement-dependent cytotoxicity assays showed that a high level of donor reactive antibodies was presented in sensitized sera. Compared with the non-sensitized recipients, the homing assay showed significantly decreased distributions of allogeneic donor BMCs in peripheral blood, spleen and femur of sensitized recipients. Non-sensitized recipients survived long term after irradiation, while all the sensitized recipients died within 12 - 15 days. Fourteen days post transplantation, the white blood cells and BMCs of non-sensitized recipients were （3240 ±300）× 10^6/L and （396±27）× 10^6/femur, respectively ; while the white blood cells and BMCs of sensitized recipients were （ 320 ± 80 ） × 10^6/L and （ 6 ± 2 ） × 10^6/femur, respectively; the differences were statistically significant between this two groups （P 〈 0.05 ）. Severn days post transplantation, the percentage of donor cells in bone marrow of non-sensitized and sensitized recipients was （48.07 ± 4.70） % and （ 0.77 ± 0.11 ） % , respectively, and the differences were statistically significant （ P 〈 0.05 ）. Furthermore, the white blood cells and BMCs following transplantation decreased along with time in sensitized recipients. The immune experiments of complement-dependent cytotoxicity reaction, cytotoxic T lymphocytes reaction and antibody-dependent cellular cytotoxicity showed the cytotoxic indexes were higher in sensitized group than the non-sensitized group. Conclusion A sensitized model was established by transfusions of allogeneic spleen cells. Allogeneic donor BMCs were rejected in sensitized recipients, and its mechanism might be through immune impairment pathways.