他克莫司对重型再生障碍性贫血患者效应T细胞抑制作用的研究  

Inhibitory effects of tacrolimus on effector T cells from patients with severe aplastic anemia

作　　者：董琪娥[1] 付蓉[1] 刘春燕[1] 阮二宝[1] 王晓明[1] 王国锦[1] 瞿文[1] 刘鸿[1] 吴玉红[1] 宋嘉[1] 邢莉民[1] 关晶[1] 李丽娟[1] 王化泉[1] 邵宗鸿[1] 

机构地区：[1]天津医科大学总医院血液科,300052

出　　处：《中华医学杂志》2013年第20期1541-1545,共5页National Medical Journal of China

基　　金：国家自然科学基金（30971286、30971285）;卫生部卫生行业科研专项（201202017）;天津市卫生局科技攻关项目（11KGl35）;天津市自然科学基金（12JczDJc21500）;中华医学会分子生物学临床应用研究专项基金（CAMB042010）

摘　　要：目的体外观察他克莫司（FK506）对重型再生障碍性贫血（SAA）患者骨髓CD8＋HLA-DR＋T细胞的抑制作用，SAA患者效应T细胞功能与临床的相关性，进一步探讨SAA的免疫发病机制。方法选取2012年2至9月天津医科大学总医院血液科收治的SAA患者16例，免疫磁珠分选患者骨髓CD8＋HLA-DR＋T细胞，分别加入不同浓度白细胞介素2（IL-2）、FK506，培养72h用光密度法测培养孔细胞增殖；用淋巴分离液分离上述SAA患者骨髓T淋巴细胞，分别加入IL-2、FKS06、环孢素（CsA），并设对照组（不加任何刺激因子），共培养18h加入佛波酯等活化4h，用流式细胞仪测CD8＋HLA-DR＋T细胞内肿瘤坏死因子β（TNF-β）蛋白表达水平；并进一步分析TNF-β表达量与外周血象及CD4＋／CD8＋T细胞比例的关系。结果当IL-2浓度≥20U／ml时，反应细胞增殖[吸光度（A）值：0．538±0．142]明显高于对照孔（0．505±0．153）（P〈0．05）；加入FKS06后，4值降低为（0．386±0．124）（P〈0．05）。IL-2组细胞内TNF-β蛋白表达（73．36％±16．73％）明显高于空白对照组（66．61％±16．20％），FK506组与FK506＋CsA组TNF-β蛋白表达（47．78％±20．09％、42．23％±21．35％）明显低于空白对照组（均P〈0．05），但是FK506＋CsA组与FK506组间差异无统计学意义（P〉0．05）。TNF-β表达量与患者外周血网织红细胞比例呈负相关，与患者外周血淋巴细胞百分比呈正相关，与患者CD4＋／CD8＋T细胞呈负相关（r=一0．86、0．77、一0．90，均P〈0．05）。结论IL＋2能促进SAA患者CD8＋HLA-DR＋T细胞增殖，FK506能抑制此增殖作用；IL-2能促进CD8＋HLA-DR＋T细胞分泌高水平的TNF-β，FK506能抑制其分泌；FK506联合CsA对CD8＋HLA-DR＋T细胞抑制作用与单用FK506作用相当。Objective To explore the inhibitory effects of tacrolimus （FK506） on effector T cells in vitro and examine the relationship between effector T cells and clinical features in patients with severe aplastic anemia （SAA） to elucidate its immune mechanism. Methods The CD8 ＋ HLA-DR ＋ cells, sorted by immunomagnetic separation from bone marrow mononuclear cells （BMMNC） of 16 SAA patients, were cultured in different concentrations of interleukin-2 （IL-2） alone or with FKS06 for 72 hours. The proliferation effect was measured with methyl thiazolyl tetrazolium （MTT） method. The T lymphocytes were sorted from the SAA patients by lymphocyte separation medium and cultured alone or with IL-2 or with FK506 or FKS06 plus cyclosporin A （ CsA ） for 18 hours. The expression of tumor necrosis factor-β （TNF-β） in CD8 ＋ HLA-DR ＋ T cells was analyzed by flow cytometry. The relationship between the expression of TNF-β and the clinical data, including percentages of reticulocyte and lymphocytes in peripheral bloodcell count and ratio of CD4 ＋ T cells and CD8 ＋ T cells, was also analyzed. Results At the concentration of IL-2 greater than or equal to 20 U/ml, the cell proliferation （A values, 0. 538 ±0. 142） were significantly higher than that in the blank culture hole （ 0. 505± 0. 153 ） （ P 〈 0. 05 ）. The A values significantly decreased（0. 386± 0. 124）after the addition of FK506 （P 〈 0.05）. Compared with control group, the expression of TNF-β was significantly higher in IL-2 group （73.36%±16.73% vs 66. 61% ±16.20% ,P 〈 0.05 ） , significantly lower in FK506 and FK506 plus CsA groups （ P 〈 0.05 ）. No significant differences existed between the FK506 and FK506 plus CsA groups （47.78% ±20. 09% and 42. 23% ±21.35%, P 〉 0. 05 ）. The expression of TNF-β in SAA was negatively correlated with the percentage of reticulocyte and the ratio of CD4＋T cell and CD8＋ T cell, positively correlated with the percentage of lymphocyte in peripheral blood count（

关 键 词：贫血 再生障碍性 肿瘤坏死因子类 CD8+HLA-DR+T细胞 他克莫司 

分 类 号：R5[医药卫生—内科学]