恶性血液病患者异基因造血干细胞移植后骨髓T淋巴细胞亚群重建的动态观察

目的 研究恶性血液病患者经异基因造血干细胞移植（allo-HSCT）后骨髓免疫重建中T淋巴细胞的重建规律及其与外周血中T淋巴细胞重建的差异。方法 本研究采用前瞻性研究设计。收集2015年9月至2017年1月在我院血液科行allo-HSCT的41例恶性血液病患者的骨髓及外周血标本，收集同期7名健康供者的骨髓及外周血标本作为对照样本。用流式细胞术分析移植前和移植后15、30、60、90、180 d时的T淋巴细胞亚群分布，包括CD4⁺T淋巴细胞、CD8⁺ T淋巴细胞、辅助性T细胞（Th）1、Th2，并用Luminex技术检测Th1相关细胞因子白细胞介素2受体（IL-2R）、白细胞介素18（IL-18）水平。结果 恶性血液病患者在移植后15 d和30 d时骨髓中CD4⁺T淋巴细胞比例均低于对照组（P均<0.05），至移植后180 d仍未见回升；CD8⁺ T淋巴细胞比例在移植后早期（15、30 d）低于对照组（P均<0.01），60 d时恢复至正常水平；骨髓中CD4⁺和CD8⁺ T淋巴细胞比例整体水平均低于外周血中水平（P=0.001、0.002）。恶性血液病患者在移植后15、30、60、90、180 d时骨髓中Th1比例均高于对照组（P均<0.05），且整体水平高于外周血中水平（P=0.006）；骨髓中Th2比例在移植后90 d内均无明显变化，在180 d时高于对照组（P=0.034），但整体水平与外周血中水平无明显差异（P>0.05）。骨髓中CD4⁺/ CD8⁺ T淋巴细胞比值在移植后逐渐下降，至移植后180 d时低于对照组（P=0.040）；而骨髓中Th1/Th2比值在移植后90 d内各时间节点（15、30、60、90 d）均高于对照组（P均<0.01），180 d时与对照组差异无统计学意义（P>0.05）。恶性血液病患者骨髓和外周血中IL-2R水平在移植后15、30、60、90 d均高于对照组（P均<0.05）；IL-18水平在移植后15、30、60 d高于对照组（P均<0.05），但移植后90 d时仅外周血中水平与对照组差异有统计学意义（P=0.021）；骨髓与外周血中IL-2R和IL-18整体水平均无明显差异（P均>0.05）。结论 恶性血液病患者allo-HSCT后，骨髓中各T淋巴细胞亚群的重建规律不同，且与外周血中有所差异。

Re-constitution of T lymphocyte subsets in bone marrow of patients with hematological malignancies after allogenic hematopoietic stem cell transplantation

Objective To explore the re-constitution rule of T lymphocyte subsets in bone marrow of patients with hematological malignancies after allogenic hematopoietic stem cell transplantation (allo-HSCT), and its differences with those in the peripheral blood. Methods This study was a prospective study. We collected the bone marrow and peripheral blood samples from 41 patients with hematological malignancies receiving allo-HSCT treatment in Department of Hematology of our hospital from Sep. 2015 to Jan. 2017. During the same period, bone marrow and peripheral blood samples of 7 healthy donors were collected as control samples. Flow cytometry was used to evaluate the distribution of T lymphocyte subsets, including CD4⁺ T cells, CD8⁺ T cells, T-helper cell (Th)1 and Th2 before transplantation and 15, 30, 60, 90 and 180 d after transplantation. Luminex technique was used to evaluate Th1-related cytokines (interleukin 2 receptorIL-2R] and interleukin 18IL-18]). Results The proportions of CD4⁺ T cells in bone marrow of the patients with hematological malignancies were significantly lower versus the healthy controls 15 and 30 d after transplantation (both P<0.05), and no recovery was found 180 d after transplantation. The proportions of CD8⁺ T cells in bone marrow of the patients with hematological malignancies were significantly lower versus the healthy controls 15 and 30 d after transplantation (both P<0.01), and it recovered to normal level 60 d after transplantation. The total proportions of CD4⁺ and CD8⁺ T lymphocytes in bone marrow were both significantly lower than those in the peripheral blood of the patients with hematological malignancies (P=0.001, 0.002). The proportions of Th1 in bone marrow of the patients with hematological malignancies were significantly higher than those of the healthy controls 15, 30, 60, 90 and 180 d after transplantation (all P<0.05), and the total level was significantly higher than that in peripheral blood (P=0.006). The proportions of Th2 in bone marrow did not change significantly within 90 d after transplantation, but it was significantly higher 180 d after transplantation than that of the healthy controls (P=0.034), and the total level was similar to the total level of peripheral blood (P>0.05). The ratio of CD4⁺/CD8⁺ T lymphocytes in bone marrow was gradually decreased after transplantation, and significantly lower versus the healthy controls 180 d after transplantation (P=0.040). The ratios of Th1/Th2 in bone marrow were significantly higher than those of the healthy controls 15, 30, 60 and 90 d after transplantation (all P<0.01), while it was similar to the healthy control level 180 d after transplantation (P>0.05). The levels of IL-2R in bone marrow and peripheral blood of the patients with hematological malignancies were significantly higher than those of healthy controls 15, 30, 60 and 90 d after allo-HSCT (all P<0.05). The levels of IL-18 were significantly higher than those of healthy controls 15, 30 and 60 d after allo-HSCT (all P<0.05), and that in peripheral blood 90 d after transplantation was also significantly different from that of healthy controls (P=0.021). There were no significant differences in IL-2R or IL-18 between bone marrow and peripheral blood (both P>0.05). Conclusion After allo-HSCT, the re-constitution rules of different T lymphocyte subsets in bone marrow of patients with hematological malignancies are different, and are different from those in peripheral blood.