中华皮肤科杂志 ›› 2018, Vol. 51 ›› Issue (9): 653-657.doi: 10.3760/cma.j.issn.0412-4030.2018.09.004

• 论著 • 上一篇    下一篇

烟曲霉对人急性单核细胞白血病细胞系产生肿瘤坏死因子α、活化信号分子p38丝裂原活化蛋白激酶的影响

童建波1,杜蕾蕾1,曾荣1,王丽玮1,刘宇甄1,段志敏1,陈青2,李岷3   

  1. 1. 中国医学科学院皮肤病研究所
    2. 江苏省血液中心
    3. 南京 中国医学科学院北京协和医学院皮肤病研究所
  • 收稿日期:2017-10-09 修回日期:2018-03-24 出版日期:2018-09-15 发布日期:2018-08-30
  • 通讯作者: 李岷 E-mail:drlimin@sina.cn
  • 基金资助:
    中国医学科学院医学与健康科技创新工程项目;国家973计划项目;国家自然科学基金;江苏省自然科学基金;江苏省十三五强卫工程项目

Effect of Aspergillus fumigatus on the of tumor necrosis factor-α and activation of intracellular signaling molecule p38 mitogen-activated protein kinase by a human acute monocytic leukemia cell line THP-1

  • Received:2017-10-09 Revised:2018-03-24 Online:2018-09-15 Published:2018-08-30
  • Supported by:
    CAMS Innovation Fund for Medical Sciences;National Basic Research Program (973 Program) of China;National Natural Science Foundation of China;Natural Science Foundation of Jiangsu Province of China;"Thirteen Five" Key Medical Talent′s Project in Science and Education of Jiangsu Province

摘要: 目的 探讨烟曲霉对人急性单核细胞白血病细胞系THP-1细胞分泌肿瘤坏死因子α(TNF-α)和细胞内信号分子p38丝裂原活化蛋白激酶(MAPK)激活的影响。方法 106、107 CFU/ml烟曲霉悬液(106和107 CFU/ml烟曲霉组)、100 mg/L阳性刺激物β-葡聚糖(β-葡聚糖组)及培养基(空白对照组)分别与2 × 105/ml THP-1细胞共孵育1、3、6 h,实时荧光定量PCR分析各组THP-1细胞TNF-α mRNA表达水平。107 CFU/ml烟曲霉悬液(烟曲霉组)、β-葡聚糖(β-葡聚糖组)及培养基分别作用THP-1细胞24 h,酶联免疫吸附法检测各组培养上清液中TNF-α含量。Western印迹法检测107 CFU/ml烟曲霉体外作用THP-1细胞后15、30、60 min p38MAPK和磷酸化p38MAPK水平。采用20 μmol/L SB203580预先与THP-1细胞共培养2 h后,再用107 CFU/ml烟曲霉、β-葡聚糖或培养基作用6 h,检测各组THP-1细胞TNF-α mRNA表达水平变化。结果 106、107 CFU/ml烟曲霉组、100 mg/L β-葡聚糖组及空白对照组TNF-α mRNA表达水平差异有统计学意义(F = 110.983,P < 0.001),且随培养时间延长,THP-1细胞TNF-α mRNA水平增高(F = 701.680,P < 0.001)。107 CFU/ml烟曲霉刺激THP-1细胞24 h后,TNF-α蛋白水平(6 236.30 ± 437.12 ng/L)较空白对照组(132.10 ± 0.61 ng/L)明显升高(P < 0.01)。107 CFU/ml烟曲霉作用THP-1细胞30 min后磷酸化p38MAPK蛋白水平明显升高,60 min时开始减弱。SB203580阻断的烟曲霉组TNF-α mRNA表达水平(3.83 ± 0.62)较未阻断的烟曲霉组(187.23 ± 21.62)明显降低。结论 人THP-1细胞体外与烟曲霉作用后激活信号分子p38MAPK并分泌TNF-α,表明单核细胞可能参与抗烟曲霉感染固有免疫反应。

关键词: 烟曲霉菌, 肿瘤坏死因子α, p38丝裂原活化蛋白激酶类, THP-1细胞

Abstract: Tong Jianbo, Du Leilei, Zeng Rong, Wang Liwei, Liu Yuzhen, Duan Zhimin, Chen Qing, Li Min Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Jiangsu Key Laboratory of Molecular Biology for Skin Diseases, Nanjing 210042, China (Tong JB, Du LL, Zeng R, Wang LW, Liu YZ, Duan ZM, Li M); Jiangsu Province Blood Center, Nanjing 210042, China (Chen Q) Corresponding authors: Li Min, Email: drlimin@sina.cn; Chen Qing, Email: chenqing90@yahoo.com 【Abstract】 Objective To evaluate the effect of Aspergillus fumigatus on the of tumor necrosis factor-α (TNF-α) and activation of intracellular signaling molecule p38 mitogen-activated protein kinase (p38MAPK) in a human acute monocytic leukemia cell line THP-1. Methods Cultured THP-l cells (2 × 105/ml) were divided into 4 groups to be treated with Aspergillus fumigatus suspensions at concentrations of 106 and 107 colony-forming units (CFU)/ml (106- and 107-CFU/ml Aspergillus fumigatus groups), 100 mg/L β-glucan (a positive stimulus, β-glucan group), culture medium (blank control group) respectively for 1, 3 and 6 hours. Real-time fluorescence-based quantitative PCR (qPCR) was conducted to determine the mRNA of TNF-α in the THP-1 cells in the above groups. Some other THP-l cells were treated with 107 CFU/ml Aspergillus fumigatus suspensions (107-CFU/ml Aspergillus fumigatus group), β-glucan (β-glucan group) and culture medium (blank control group) separately for 24 hours, and enzyme-linked immunosorbent assay (ELISA) was performed to detect the level of TNF-α in the culture supernatant of THP-1 cells. Western blot analysis was conducted to detect the levels of p38MAPK and phosphorylated p38MAPK in THP-1 cells after 15-, 30- and 60-minute treatment with 107 CFU/ml Aspergillus fumigatus suspensions. After 2-hour incubation with the p38MAPK inhibitor SB203580 (20 μmol/L), some THP-1 cells were additionally treated with 107 CFU/ml Aspergillus fumigatus suspensions, β-glucan and culture medium separately for 6 hours, and those without SB203580 treatment served as the control group. Then, qPCR was performed to measure the mRNA of TNF-α in the THP-1 cells in the above groups. Results The mRNA of TNF-α significantly differed among the 106- and 107-CFU/ml Aspergillus fumigatus groups, β-glucan group and blank control group (F = 110.983, P < 0.001), and significantly increased over time (F = 701.680, P < 0.001). After 24-hour treatment with 107 CFU/ml Aspergillus fumigatus suspensions, the TNF-α level(6 236.30 ± 437.12 ng/L)significantly increased compared with the blank control group (132.10 ± 0.61 ng/L, P < 0.01). Thirty minutes after the treatment with 107 CFU/ml Aspergillus fumigatus suspensions, the phosphorylated p38MAPK level significantly increased, but started to decrease at 60 minutes. The mRNA of TNF-α was significantly lower in the SB203580-treated Aspergillus fumigatus groups (3.83 ± 0.62) than in the SB203580-untreated Aspergillus fumigatus groups (187.23 ± 21.62). Conclusion After the treatment with Aspergillus fumigatus, human THP-1 cells can activate the signal molecule p38MAPK and secrete TNF-α, suggesting that monocytes may participate in the innate immune response to Aspergillus fumigatus infection.

Key words: Aspergillus fumigatus, Tumor necrosis factor-alpha, p38 Mitogen-activated protein kinases, THP-1 cells