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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 investigate the effect of tumor necrosis factor-β (TNF-β) on the hematopoietic activity of effector T cells (CD8 + HLA-DR + T cells) in patients with severe aplastic anemia (SAA) In the role. Methods: Bone marrow CD8 + HLA-DR + T cells from 15 SAA patients were selected as effector cells by immunomagnetic beads. Negatively sorted CD3 + T cells from 15 normal human bone marrow mononuclear cells as target cells. Were mixed and cultured in the system were added different concentrations of TNF-β (final concentrations were 0,15,25,50 ng / ml) for the experimental group, and set the blank control group, incubated for 72 hours, by flow cytometry The apoptosis of target cells was detected by Annexin V, and the levels of IL-10 and IFN-γ in culture supernatants were determined by ELISA. Results: The percentage of apoptotic cells in the blank control group and the three experimental groups were (49.85 ± 20.33)%, (51.65 ± 20.34)%, (55.94 ± 20.19)% and (57.72 ± 19.45)%, respectively. The final concentration of TNF- 50 ng / ml group and 25 ng / ml group were significantly higher than the blank group and 15 ng / ml group (P <0.05), but the blank group and 15 ng / ml group, 25 ng / ml group and 50 There was no significant difference in the percentage of apoptotic cells between the two groups (P> 0.05). The concentrations of IL-10 in the culture supernatant of the blank control group and experimental groups were (217.99 ± 29.47) ng / ml, (216.47 ± 29.00) ng / ml, (212.15 ± 13.19) ng / ml, there was no significant difference among the groups (P> 0.05). The level of IFN-γin the culture supernatants with the final concentration of 50 ng / ml TNF-β [(593.50 ± 48.18) ng / ml] was significantly higher than that in the blank control group [(544.85 ± 69.77) ng / ml] (567.38 ± 74.51) ng / ml], 25 ng / ml group [(544.05 ± 79.69) ng / ml] (P <0.05). Conclusions: TNF-β can enhance the apoptosis of bone marrow hematopoietic cells induced by effector T cells in SAA patients in a concentration-dependent manner. High levels of TNF-β may also promote the secretion of IFN-γ by CD8 + HLA-DR + T cells in SAA patients Has a synergistic cytotoxic effect.