肿瘤坏死因子-β对重型再生障碍性贫血患者效应T细胞损伤骨髓造血的影响

Effects of TNF-β on damage of hematopoiesis by CD8^+HLA-DR^+T cells of SAA patients

目的:研究肿瘤坏死因子-β(TNF-β)对重型再生障碍性贫血(SAA)患者效应T细胞(CD8+HLA-DR+T细胞)损伤骨髓造血的影响,探讨TNF-β在SAA免疫发病中的作用。方法:以免疫磁珠阳性分选15例SAA患者骨髓CD8+HLA-DR+T细胞作为效应细胞,以阴性分选去除15名正常人骨髓单个核细胞中CD3+T细胞后作为靶细胞,两者混合培养,于体系中分别加入不同浓度TNF-β(终浓度分别为0、15、25、50 ng/ml)为实验组,并设空白对照组,孵育72小时后,通过流式细胞术以Annexin V检测靶细胞凋亡状况,采用ELISA法测定培养上清液IL-10、IFN-γ水平。结果:空白对照组及3个实验组细胞凋亡比例分别为(49.85±20.33)%、(51.65±20.34)%、(55.94±20.19)%、(57.72±19.45)%,TNF-β终浓度为50 ng/ml组及25 ng/ml组均明显高于空白组和15 ng/ml组细胞凋亡比例(P<0.05),但空白组与15 ng/ml组,25 ng/ml组与50 ng/ml组细胞凋亡比例无显著差异(P>0.05)。空白对照组及各实验组培养上清IL-10浓度分别为(217.99±29.47)ng/ml、(216.47±29.00)ng/ml、(212.15±13.19)ng/ml、(218.01±28.61)ng/ml,各组差异无统计学意义(P>0.05)。TNF-β终浓度50 ng/ml组培养上清IFN-γ水平[(593.50±48.18)ng/ml]明显高于空白对照组[(544.85±69.77)ng/ml],15 ng/ml组[(567.38±74.51)ng/ml]、25 ng/ml组[(544.05±79.69)ng/ml](P<0.05)。结论:TNF-β能增强SAA患者效应T细胞诱导骨髓造血细胞凋亡,并呈浓度依赖性;高水平的TNF-β可能还促进SAA患者CD8+HLA-DR+T细胞分泌IFN-γ,两者具有协同细胞毒作用。

Objective：To explore the effects of tumor necrosis factor-β（TNF-β）on damage of hematopoietic cells by CD8＋HLA-DR＋ effector T cells of the patients with severe aplastic anemia（SAA）,and to prove the role of TNF-β in immunopathogenesis of SAA.Methods：CD8＋HLA-DR＋ T cells as effector cells were sorted from bone marrow mononuclear cells（BMMNC） of SAA patients by magnetic activated cell sorting system（MACS）.CD3＋ cells MACS depleted BMMNC of normal controls as target cells were co-cultured with the effector cells（1∶1） in different concentrations of TNF-β（0,15,25 and 50 ng/ml） for 72 hours.The percentage of Annexin V positive cells were measured by flow cytometry.The quantitive assay of IL-10,IFN-γ in the culture supernatant was also performed using the enzyme linked immunosorbent assay kit.Results：The apoptotic rate（AR） of target cells in control group and experimental groups were（49.85±20.33）%,（51.65 ±20.34）%,（55.94 ±20.19）% and（57.72 ±19.45）%.AR of target cells in 25 ng/ml and 50 ng/ml TNF-β groups were significiantly higher than those in control group and 15 ng/ml TNF-β group（P〈0.05）.There were no significiant difference between those in control group and 15 ng/ml TNF-β group,also between those in 50 ng/ml and 25 ng/ml TNF-β groups（all P〉0.05）.The levels of IL-10 in the culture supernatant in different groups were（217.99±29.47）ng/ml,（216.47±29.00）ng/ml,（212.15±13.19）ng/ml and（218.01±28.61）ng/ml.There were no difference among them（P〉0.05）.The levels of IFN-γ in the culture supernatant of 50 ng/ml TNF-β group[（593.50±48.18）ng/ml] was significiantly higher than those of control group [（544.85±69.77）ng/ml],15 ng/ml TNF-β group [（567.38±74.51）ng/ml] and 25 ng/ml TNF-β group [（544.05±79.69）ng/ml]（P〈0.05）.Conclusion：TNF-β could enhance the apoptosis of hematopoietic cells induced by effector T cells of SAA patients in dose-respanse manner.High level of TNF-β might also promote CD8＋HLA-DR＋T cell secrete IFN-γ which could collaborative by damage hematopoiesis.