药物去势与异基因造血干细胞移植后胸腺损伤关系的实验研究

Study on relationship between chemical castration and thymic function following hematopoietic stem cell transplantation

目的探讨促黄体激素释放激素（LHRH）去势对异基因造血干细胞移植（allo—HSCT）后小鼠胸腺功能的保护作用。方法构建C57BL／6→BALB／c小鼠MHC不相合allo—HSCT模型；对急性移植物抗宿主病（aGVHD）严重程度评分；采用蛋白液相芯片技术检测小鼠胸腺细胞内IFNγ、TNFα和IL-1β表达水平，实时定量PCR分析小鼠外周血T细胞受体重排删除环（sjTREC）水平。结果单纯allo—HSCT组（A组）、LHRH处理allo—HSCT组（B组）、同基因移植组（C组）三组小鼠均获得造血重建，WBC〉1．0×10^9／L时间分别为（11．2±1．4）、（9．8±0．6）和（9．7±0．7）d（P=0．003）。A、B两组aGVHD出现时间分别为（11．4±1．2）d和（14．1±0．7）d（P=0．000），C组未发生aGVHD，A、B组aGVHD发生率为100％。A组和B组aGVHD评分分别为（9．1±0．7）和（5．1±1．0）分（P=0．000）。正常受鼠胸腺细胞内IFNγ、TNFα和IL-1β水平分别为（2．3±2．5）、（1．7±1．1）和（1．8±1．2）pg／ml。A、B、C三组IFNγ水平分别为（10．5±2．1）、（6．7±2．1）和（5．2±3．3）ng／ml；TNFα水平分别为（7．0±2．6）、（4．3±0．8）和（3．0±1．8）pg／ml；IL-1β水平分别为（24．9±9．0）、（17．4±3．9）和（10．8±3．1）pg／ml。A、B组IFM、TNFd和IL-1β水平比正常受鼠组明显升高（P值均〈0．01）；C组IFNγ和IL-1β水平明显高于正常对照组（P值分别为0．015，0．013）。A组IFNγ、TNFα和IL-1β水平比B组明显升高（P值分别为0．002、0．002、0．004）；A组IFNγ、TNFα和IL-1β水平较C组明显升高（P值均为0．000）。Linear Regression分析提示，IFNγ和TNFα水平越高，aGVHD评分越高（r2=0．359，P=0．05；r2=0．228，P=0．019）。正常受鼠1000个外周血单个核细胞中sjTREC拷贝数为（39．4±44．7）。A、B和C组小鼠分别为（12．3±13．0）、（58．0±71．8）和（19．6±14．6），与正常受鼠组sjTRECs水平差异无统计学意义。结论细胞因子IFNγ、TNFα和IL-1β参与了aGVHD对胸腺的损伤过程；LHRH药物去势能减轻aGVHD对胸腺的损伤，并通过保护胸腺减轻aGVHD的严重程度，其机制可能与降低胸腺细胞内IFNγ、TNFα水平有关。

Objective To evaluate the impact of luteinizing hormone-releasing hormone （LHRH） on the protection of thymic function after allogenic hematopoietie stem cell transplantation （allo-HSCT）. Methods Murine model of MHC mismatched allogeneic HSCT （C57BL/6→BALB/c） was established. The severity of acute graft-versus-host-disease （GVHD） was assessed according to a clinical scoring system. The intra-cellular levels of IFNγ、TNFα and IL-1β in thymoeyte were analyzed by protein array and thymic function by quantification of signal-joint TCR rearrangement excision circles （sjTRECs）. Results All recipients in group A （allogeneic mice）, B （ allogeneic LHRH castrated-mice） and C （syngenic mice） achieved hematopoietic reconstitution. White blood cell （ WBC ） over 1. 0 × 10^9/L in groups A, B and C were on day （11.2±1.4）, day （9.8±0.6） and day （9.7±0.7）, respectively （P=0.003, 0.002）. The onset of acute GVHD in group B was （ 14.1 ± 0.7 ） d and in group A was （ 11.4 ± 1.2） d （ P = 0.000）. All mice in groups A and B developed acute GVHD. No mice occurred aGVHD in group C. The average scores of acute GVHD in groups A and B were （9.1 ± 0.7 ） and （5.1 ± 1.0）, respectively （ P = 0. 000）. The levels of IFN3,, TNFα and IL-1β in control group were （2.3 ± 2.5 ） ng/ml, （ 1.7 ± 1.1 ） pg/ml and （ 1.8 ± 1.2 ） pg/ml, respectively. The IFNγ levels in groups A, B and C were （ 10.5 ± 2.1 ） ng/ml, （6.7 ± 2.1 ） ng/ml and （5.2 ±3.3） ng/ml, TNFα levels were （7.0 ±2.6） pg/ml, （4.3 ±0.8） pg/ml and （3.0 ± 1.8） pg/ ml, and IL-1β levels were （24.9 ±9.0） pg/ml, （17.4 ±3.9） pg/ml and （10.8 ±3.1） pg/ml, respectively. There were significant differences in the levels of cytokines between group A and the control group （ P = 0.000, 0. 000, 0. 000）. The levels of cytokines in group B were significantly higher than those in control group （P =0. 000,0. 003,0. 000）. The levels of IFNγ and IL-β in group C were significantly higher than those of in control group （P = 0. 015, 0. 013 ） , and so did in group A than in group B （P = 0. 002, 0. 002, 0.004） , and in group A than in group C （P = 0.000, 0. 000, 0. 000）. The analysis of linear regression showed that the average levels of IFNγand TNFα paralled with aGVHD scores （ r2 = 0. 359, P = 0. 045 ; r2 = 0. 228, P =0.019）. The average sjTRECs copies/lO00 PBMNCs were （39.4 ± 44.7） in the control group and （ 12.3 ± 13.0）, （58.0 ±71.8） and （ 19.6 ± 14.6） in groups A, B and C, respectively. There was no significant difference in the multiple comparisons of peripheral blood levels of sjTRECs among these four groups （ P = 0. 468）. Conclusion IFNγ ,TNFα and IL-1β might be involved in the damage to the thymus by acute GVHD. Sex steroid inhibitor can not only reduce the severity of thymic damage after allo-HSCT, but also reduce the severity of aGVHD and the mechanism might be associated with the reduction of intra-cellular levels of IFNγ in thymocyte.