肿瘤坏死因子α对喉黏膜间充质干细胞免疫调节能力的影响

Influence of TNF-α on the immunomodulatory property of laryngeal mucosa mesenchymal stromal cells′

目的研究肿瘤坏死因子α(tumor necrosis factor-alpha,TNF-α)对喉黏膜间充质干细胞(laryngeal mucosa mesenchymal stromal cell,LM-MSC)免疫调节能力的影响及其潜在的分子机制。方法2017年4月至2018年3月,取喉癌全切手术患者组织标本中的正常喉黏膜组织,通过酶消化法分离培养LM-MSC。通过LM-MSC细胞与T细胞体外直接共培养模型,检测LM-MSC的免疫调节能力。通过炎症因子TNF-α长期刺激在体外模拟慢性炎症模型,比较正常与慢性刺激后的LM-MSC免疫调节能力的差异。通过Western blot和实时定量反转录PCR(RT-qPCR)等方法检测正常与TNF-α慢性刺激后的LM-MSC表观遗传基因组蛋白乙酰基转移酶复合体(general control non-repressed protein5,GCN5)的表达差异。采用SPSS 17.0统计学软件对数据进行统计分析。结果在炎症因子TNF-α慢性作用下,与对照组(设为1)相比,GCN5的RNA相对表达量为0.31±0.03(3 d)和0.53±0.06(7 d),差异有统计学意义(F=13.45,P<0.05),LM-MSC诱导T细胞凋亡率低于对照组,差异有统计学意义[6.27%±0.81%(3 d)和4.99%±0.52%(7 d)比10.02%±1.02%,F=11.13,P<0.05]。并且LM-MSC诱导T细胞凋亡的能力受到GCN5的调节。在TNF-α的慢性炎症刺激后,FAS/FASL与GCN5表达水平一致,并且随着GCN5的变化而变化。结论TNF-α慢性刺激后,LM-MSC中GCN5的表达降低,从而使得其免疫调节能力受损。

Objective To investigate the effect of tumor necrosis factor-alpha(TNF-α)on the immunoregulatory capacity of laryngeal mucosal mesenchymal stromal cells (LM-MSCs) and its potential molecular mechanism, and provide a theoretical basis for the study of chronic laryngitis. Methods LM-MSCs were separated from epiglottal mucosa. The LM-MSCs cells were directly co-cultured with T cells in vitro to detect the immunomodulatory property of LM-MSCs. After long-term stimulation with inflammatory factors TNF-α in vitro, the differences were compared in the immunomodulatory ability of LM-MSCs between normal LM-MSCs and TNF-α stimulated LM-MSCs. The expression of general control non-repressed protein5(GCN5), FAS, FASL in normal LM-MSCs and TNF-α stimulated LM-MSCs was detected by Western blot and quantitative real-time RT-PCR(RT-qPCR). Results After chronic stimulation of TNF-α, the RNA relative expression of GCN5 was 0.31±0.03 (3 days) and 0.53±0.06 (7 days) compared with control group, showing significant difference (F=13.45, P<0.05). The percentage of LM-MSC-induced T cell apoptosis was 6.27%±0.81%(3 days) and 4.99%±0.52%(7 days) in chronic stimulation group compared with control group 10.02%±1.02%. There is a significant difference among these groups (F=11.13, P<0.05). Moreover, the ability of LM-MSCs to induce T cell apoptosis is regulated by GCN5. Conclusion With the chronic stimulation of TNF-α, the expression of GCN5 in LM-MSCs is decreased, thus impairing its immunoregulatory capacity.