小鼠异基因造血干细胞移植后使用G-CSF对aGVHD的影响

Effect of granulocyte colony-stimulating-factor on acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation in a murine model

目的：探讨小鼠异基因造血干细胞移植（allo-HSCT）后使用粒细胞集落刺激因子（G-CSF）对急性移植物抗宿主病（a GVHD）的影响及其可能的机制。方法：以雄性C57BL/6小鼠（H-2b）为异基因供鼠,雄性BALB/c小鼠（H-2d）为同基因供鼠;以接受8 Gy[60Co]γ射线全身照射（TBI）预处理雌性BALB/c小鼠为受鼠,并随机分为7组：单纯TBI组、同基因骨髓＋脾细胞移植（Syn-BMST）组、异基因骨髓移植（allo-BMT）组、异基因骨髓＋脾细胞移植（allo-BMST）组、Syn-BMST后G-CSF给药（Syn-BMST＋G-CSF）组、allo-BMT后G-CSF给药（allo-BMT＋G-CSF）组和allo-BMST后G-CSF给药（allo-BMST＋G-CSF）组,各G-CSF给药组从移植后第1天（＋1 d）开始皮下注射G-CSF 2μg/d,观察至＋60 d。比较各组生存时间、a GVHD发生情况和病理改变,流式细胞术检测骨髓H2-Kb＋细胞百分率（异基因嵌合率）,比较allo-BMST和allo-BMST＋G-CSF组＋10 d时血清细胞因子（IL-2、IL-4、IFN-γ和TNF-α）水平、脾总有核细胞数（Sp TNC）和脾细胞免疫表型的差异。结果：单纯TBI组小鼠于照射后9~15 d死于造血衰竭,其余各组＋10 d时均100%获得造血重建,随机抽取2只异基因骨髓移植受鼠,＋30 d供者细胞嵌合率分别为99.8%和99.4%,表明清髓性allo-HSCT模型建立成功。Syn-BMST、Syn-BMST＋G-CSF、allo-BMT和alloBMT＋G-CSF组小鼠观察至＋60 d均未发生a GVHD。与allo-BMST组相比,allo-BMST＋G-CSF组受鼠出现a GVHD时间早、程度重、病理改变严重、存活时间明显缩短（P〈0.05）、＋10 d Sp TNC明显增加（P〈0.05）、脾脏NK细胞显著扩增（P〈0.01）、DC1/DC2比值减低（P〈0.05）,而2组血清IL-2、IL-4、IFN-γ和TNF-α水平差异无统计学意义。结论：移植后使用G-CSF对小鼠异基因单纯骨髓移植后a GVHD无明显影响,但能显著加重allo-BMST后a GVHD的严重程度并缩短受鼠生存时间,该效应可能与G-CSF诱导供鼠NK细胞扩增有关,提示临床allo-HSCT后早期使用G-CSF可能触发或加重a GVHD的风险。

AIM： To explore the impact of granulocyte colony-stimulating factor（ G-CSF） on acute graft-versus-host disease（ a GVHD） after allogeneic hematopoietic stem cell transplantation（ allo-HSCT） in a murine model and its possible mechanisms. METHODS： Male C57 BL /6（ H-2b） and BALB/c（ H-2d） mice were used as the allogeneic and syngeneic donor mice,respectively. Moreover,female BALB / c mice were used as recipient mice. The recipient mice were conditioned by a single dose（ 8 Gy） of total body irradiation（ TBI）. The recipient mice were randomly divided into 7groups： TBI group,Syn-BMST control group,post-Syn-BMST G-CSF administration（ Syn-BMST ＋ G-CSF） group,alloBMT control group,post-allo-BMT G-CSF administration（ allo-BMT ＋ G-CSF） group,allo-BMST control group and post-allo-BMST G-CSF administration（ allo-BMST ＋ G-CSF） group. The mice in control groups and G-CSF administration groups were subcutaneous injected with 0. 1 m L normal saline（ NS） and 0. 1 m L NS containing 2 μg G-CSF per day from 1st day,respectively. The effect of G-CSF on a GVHD was evaluated by clinical manifestations and pathological changes,as well as survival time of the mice in different groups. The serum levels of IL-2,IL-4,IFN-γ and TNF-α in allo-BMST and alloBMST ＋ G-CSF groups were detected by ELISA at 10 th day. Flow cytometry was used to analyze the immunophenotypes of splenocytes at 10 th day. RESULTS： The mice in TBI group were all died for hematologic failure on 9 ~ 15 d after TBI. No effect of G-CSF on the survival of the mice underwent Syn-BMST and transplantation of single allogeneic marrow cells was observed. The mean survival days in allo-BMST group and allo-BMST ＋ G-CSF group were（ 34. 8 ± 4. 5） d and（ 19. 8 ±6. 1） d＇respectively（ P〈0. 01）. Moreover,post-transplant administration of G-CSF increased the spleen total nucleated cells count（ Sp TNC）,NK cells subset,and DC1 / DC2 ratio in the spleen with over 99% of donor chimerism rate at 10 th day. No difference in the levels of serum IL-2,IL-4,IFN-γ and TNF-α between the 2 group at 10 th day was found. CONCLUSION： The administration of G-CSF after allo-BMST significantly aggravates mouse a GVHD. The expansion of NK cells stimulated by G-CSF may be involved in the mechanism of generating alloreactivity against host cells. These results imply there may be potential risk of evoking or aggravating acute GVHD if G-CSF is administered in the early stage of clinical allo-HSCT.