重型再生障碍性贫血患者CD8^＋HLA-DR^＋效应T细胞调控因素的研究

Research of regulative factors on CD8^＋ HLA-DR^＋ effector T cells in severe aplastic anemia

目的体外研究重型再生障碍性贫血（SAA）患者骨髓CD8^＋HLA-R^＋6T细胞的调控因素，进一步探讨该群细胞在SAA免疫发病中的地位。方法选取2011年7月至2012年3月收治的13例初治SAA患者。免疫磁珠分选SAA患者骨髓CD8^＋HLA-DR^＋T细胞后分为3组：白细胞介素2（IL-2）组（加入IL-2终浓度分别为0、0．1、1、10、100、1000U／L）；环孢素A（CsA）组（在IL-2浓度基础上每孔加人终浓度为400ng／ml的CsA）；IL-2受体拮抗剂组（在IL-2浓度基础上每孔加入终浓度为8μg／mlIL-2受体拮抗剂），孵育72h后四甲基偶氮唑盐（MTT）法检测细胞增殖情况。Ficoil法分离SAA患者的骨髓单个核细胞后分为3组：CsA组（加入终浓度为400ng／ml的CsA）、IL-2组（加入终浓度为100U／ml的IL-2）和对照组（不加任何刺激物），孵育18h后加人佛波酯等活化4h，流式细胞仪检测CD8^＋HLA-DR^＋T细胞胞内肿瘤坏死因子B（TNF-β）蛋白的表达水平。结果IL-2浓度为10、100、1000U／L时IL．2组CD8^＋HLA-DR^＋T细胞增殖[吸光度（A）值]分别为0．36±0．12、0．41±O．12、0．46±0．14，明显高于未加IL-2的空白对照（0．23±0．11），亦高于同等IL-2浓度下的CsA组（0．18±0．05、0．19±0．00、0．20±0．04）和IL-2受体拮抗剂组（0．18±0．05、0．17±0．04、0．18±0．03，均P〈0．05）；而CsA组和IL-2受体拮抗剂组间差异无统计学意义（P〉0．05）。IL-2组CD8^＋HLA-DR^＋T细胞胞内TNF-β蛋白表达（64％±25％）明显高于对照组（46％±22％）；CsA组（27％±20％）明显低于对照组（均P〈0．05）。结论IL-2不仅显著刺激SAA患者CD8^＋HLA-DR^＋T细胞体外增殖，还可促进该群细胞分泌高水平的TNF-β，而CsA和IL-2受体拮抗剂不仅能抑制此增殖作用，同时CsA亦能显著抑制CD8^＋HLA-DR^＋T细胞分泌TNF-β。

Objective To explore the regulative factors on CD8^± HLA-DR^± T cells in the patients with severe aplastie anemia （SAA） and examine the roles of these cells in the immunopathogenesis of SAA. Methods CD8^± HLA-DR^± T cells were sorted from bone marrow mononuclear cells of 13 SAA patients from July 2011 to March 2012 by magnetic activated cell sorting system and were divided into 3 groups： interleukin 2 （IL-2） group （0, 0. 1, 1, 10, 100 and 1000 U/ml）, eyelosporine A （CsA） group （addition of 400 ng/ml CsA in each IL-2-eontaining well）, receptor antagonist group （addition of IL-2 receptor antagonist 8 μg/ml in each IL-2-containing well）. Then cell proliferation rate was evaluated by MTY assayafter a 72-hour culturing. Bone marrow mononuclear cells of the SAA patients were divided into CsA group, IL-2 group and control group and cultured for 18 hours and another 4 hours following the dosing of phorbol ester. The expression of tumor necrosis factor 13 （TNF-13） in CD8^± HLA-DR^± T cells was analyzed by flow cytometry. Results The cell proliferations of IL-2 wells at the concentrations of 10, 100 and 1000 U/L （0. 36 ± 0. 12, 0. 41 ± 0. 12, 0. 46 ± 0. 14） were significantly higher than those of the control wells （0. 23 ± 0. 11 ）, C sA group （0. 18 ± 0. 05, 0. 19 ± 0. 00, 0. 20 ± 0. 04） and receptor antagonist group （0. 18 ± 0. 05, 0. 17 ± 0. 04, 0. 18 ± 0. 03, all P 〈 0.05）. No statistic difference existed between CsA and receptor antagonist groups （P 〉 0. 05）. The expressions of TNF-β of CD8^± HLA-DR^± T ceils of the IL-2 group were higher than those of the control group （64% ±25% vs 46% ±22% ） whereas the CsA group （27% ±20% ） were lower than those of the control group （both P 〈 0. 05）. Conclusions IL-2 can significantly stimulate the proliferation of CD8^± HLA-DR^± T cells and accelerate the in vitro secretion of TNF-β in SAA patients. The proliferation may be inhibited by CsA and receptor antagonist. And the expression of TNF-β is suppressed significantly by CsA.