摘要
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.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2012年第6期544-547,共4页
Chinese Journal of Immunology
基金
国家自然科学基金(30971286
30971285)
高等学校博士学科点专项科研基金(200800620004)
天津市应用基础及前沿技术研究计划(08JCYBJC07800
09JCYBJC11200)
卫生部卫生行业科研专项(201002024)
天津市卫生局科技攻关项目(11KG135)