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目的:明确申克孢子丝菌酵母相对人急性单核细胞白血病细胞(THP-1)NF-κB信号通路激活和肿瘤坏死因子α(TNF-α)分泌的影响。方法:实时荧光定量PCR分析申克孢子丝菌酵母相刺激THP-1细胞TNF-αmRNA的表达,酶联免疫吸附法检测TNF-α分泌量,免疫印迹法分析申克孢子丝菌酵母相体外作用于THP-1细胞后IκBα和磷酸化IκBα的水平,免疫荧光法观察NF-κB-p65核转位。同时设置地塞米松(NF-κB抑制剂)抑制组,并检测100 n M地塞米松预处理THP-1细胞后TNF-αmRNA水平的变化。结果:申克孢子丝菌酵母相刺激THP-1细胞后6 hTNF-αmRNA水平显著高于空白对照组(P<0.001)。申克孢子丝菌酵母相刺激THP-1细胞后24 h,TNF-α蛋白水平为(4610.419±121.501)pg/mL,显著高于空白对照组(186.964±98.073)pg/m L,差异具有统计学意义(P<0.001)。申克孢子丝菌酵母相作用THP-1细胞后30~60 min,磷酸化IκBα蛋白水平显著升高,为时间依赖性。申克孢子丝菌酵母相组细胞核内NF-κB-p65较空白对照组荧光强度增强。100 nM地塞米松预处理各组THP-1细胞后,TNF-αmRNA水平较前明显降低。结论:人THP-1细胞体外与申克孢子丝菌酵母相作用后激活NF-κB信号通路并上调TNF-α分泌。
Objective: To clarify the effect of TNF-α secretion and activation of THP-1 NF-κB signaling pathway in S. conidia yeast cells. Methods: The expression of TNF-α mRNA in THP-1 cells stimulated by S. schenckerii was detected by real-time fluorescence quantitative PCR. The secretion of TNF-α was detected by enzyme-linked immunosorbent assay (ELISA) The levels of IκBα and phosphorylated IκBα in THP-1 cells were detected by immunofluorescence and the nuclear translocation of NF-κB-p65 was observed. At the same time, dexamethasone (NF-κB inhibitor) inhibition group was set up, and the changes of TNF-αmRNA level in THP-1 cells pretreated with 100 nM dexamethasone were measured. Results: The THNF-αmRNA level in THP-1 cells stimulated by S. cerevisiae at 6 h was significantly higher than that in the blank control group (P <0.001). The TNF-α protein level of THP-1 cells was (4610.419 ± 121.501) pg / mL 24 hours after spores were stimulated by S. schenckii, which was significantly higher than that of the blank control group (186.964 ± 98.073) pg / m L Significance (P <0.001). From 30 to 60 min after the S. cerevisiae yeast cells treated with THP-1 cells, the phosphorylated IκBα protein level was significantly increased in a time-dependent manner. The fluorescence intensity of NF-κB-p65 in nucleus of the nucleus of the conidia yeast of Schenck Syphilis increased more than that of the blank control group. After pretreatment with 100 nM of dexamethasone for 3 weeks, the levels of TNF-αmRNA in THP-1 cells were significantly decreased compared with the pre-treatment. CONCLUSIONS: Human THP-1 cells activate NF-κB signaling and up-regulate TNF-α secretion in vitro after challenge with S. conidia yeast.