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  

Effect of Aspergillus fumigatus on the expression 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

作　　者：童建波 杜蕾蕾[1] 曾荣[1] 王丽玮[1] 刘宇甄 段志敏[1] 陈青[2] 李岷[2] 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)

机构地区：[1]中国医学科学院北京协和医学院皮肤病研究所江苏省皮肤性病分子生物学重点实验室,南京210042 [2]江苏省血液中心,南京210042

出　　处：《中华皮肤科杂志》2018年第9期653-657,共5页Chinese Journal of Dermatology

基　　金：国家自然科学基金（81502739）;国家973计划项目（2013CB531600）;江苏省自然科学基金（BK20150068）;江苏省十三五强卫工程项目（ZDRCB2016010）;中国医学科学院医学与健康科技创新工程项目（2017-12M-1-017）

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仪（TNF-α）和细胞内信号分子β38丝裂原活化蛋白激酶（MAPK）激活的影响。方法10^6、10^7CFU／ml烟曲霉悬液（10^6和10^7CFU／ml烟曲霉组）、100mg／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2×10^5／mlTHP-1细胞共孵育1、3、6h，实时荧光定量PCR分析各组THP-1细胞TNF-α mRNA表达水平。10^7CFU／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24h，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10^7CFU／ml烟曲霉体外作用THP-1细胞后15、30、60minβ38MAPK和磷酸化β38MAPK水平。采用20μmol／LSB203580预先与THP-1细胞共培养2h后，再用10^7CFU／ml烟曲霉、β-葡聚糖或培养基作用6h，检测各组THP-1细胞TNF-α mRNA表达水平变化。结果10^6、10^7CFU／ml烟曲霉组、100mg／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）。1^7CFU／ml烟曲霉刺激THP-1细胞24h后，TNF-α蛋白水平（6236．30±437．12ng／L）较空白对照组（132．10±0．61ng／L）明显升高（P〈0．01）。10^7CFU／ml烟曲霉作用THP-1细胞30min后磷酸化β38MAPK蛋白水平明显升高，60rain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β38MAPK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。Objective To evaluate the effect of Aspergillus fumigatus on the expression of tumor necrosis faetor-oL （TNF-cβ） and activation of intraeellular signaling molecule p38 mitogen-activated protein kinase （p38MAPK） in a human acute monoeytic leukemia cell line THP- 1. Methods Cultured THP-1 cells （2 x 105/ml） were divided into 4 groups to be treated with Aspergillus fumigatus suspensions at concentrations of 10s and 107 colony-forming units （CFU）/ml （106- and 107-CFU/ml Aspergillusfumigatus 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 expression of TNF-ct in the THP-1 cells in the above groups. Some other THP-1 ceils were treated with 107 CFU/ml Aspergillusfumigatus suspensions （ 107-CFU/ml Aspergillusfumigatus group）, β-glucan （13-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-cβ 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 Aspergillusfumigatus suspensions. After 2-hour incubation with the p38MAPK inhibitor SB203580 （20 txmol/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 expression of TNF-ct in the THP-1 cells in the above groups. Results The mRNA expression of TNF-ct significantly differed among the 106- and lff-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）. A

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 

分 类 号：R733.7[医药卫生—肿瘤]