分枝杆菌感染肉芽肿体外模型的建立和验证  

Development and validation of an in vitro model of mycobacterial granuloma

作　　者：田蔚蔚[1] 张晓东[1] 王秋玲[1] 唐美育[1] 沈建平[1] 王洪生[1] 王千秋[1] 

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,南京210042

出　　处：《中华皮肤科杂志》2014年第3期186-191,共6页Chinese Journal of Dermatology

基　　金：国家自然科学基金（30972651）;北京协和医学院研究生创新基金（2011-1002-030）

摘　　要：目的构建可用于分枝杆菌感染肉芽肿研究的体外模型。方法采用人免疫细胞分别与分枝杆菌（海鱼杆菌、结核杆菌、卡介苗及麻风杆菌）共培养的方法构建分枝杆菌感染肉芽肿体外模型，显微镜下动态观察肉芽肿的体外形成过程，并用流式细胞仪检测不同时期细胞表面抗原分子表达，实时定量PCR法检测细胞因子的mRNA表达及ELISA法检测重要细胞因子的分泌等。结果共培养7～9d后观察到与肉芽肿结构相似的细胞聚集团块，部分细胞形成多核巨细胞；CD14、CD68、CD80等巨噬细胞表面抗原在不同的分枝杆菌组均有阳性表达；肿瘤坏死因子α、干扰素儿白介素1β、白介素10亦有不同程度的mRNA表达及分泌。结论初步构建了人分枝杆菌肉芽肿体外模型，为研究分枝杆菌感染过程中肉芽肿形成和免疫反应诱导提供条件。Objective To establish an in vitro model of mycobacterial granuloma. Methods Mononuclear ceils were isolated from peripheral blood of healthy human subjects, and stimulated to differentiate into macrophages, which were then classified into four groups to be cocultured with Myeobacteffum marinum, Mycobacterium tuberculosis, Bacillus Calmette-Gu^rin, and Mycobactegum leprae, respectively, for five days followed by incubation with peripheral blood mononuclear cells （PBMCs） from the corresponding donors to establish an in vitro model of myeobacterial granuloma. The macrophages cocuhured with PBMCs or mycobacteria alone served as the control. Microscopy was performed to dynamically visualize the formation of granuloma in vitro, flow cytometry to detect the expressions of cell surface antigens at different stages, real-time quantitative PCR and enzyme-linked immunosorbent assay （ELISA） to determine the mRNA expressions of important cytokines and their protein levels in the supernatant of macrophages, respectively. Results After 7 - 9 days of coculture with mycobacteria and PBMCs, the maerophages aggregated to form granuloma-like clumps, and some cells fused to form muhinuclear giant cells, along with the expressions of some surface antigens such as CD14, CD68 and CD86 on these macrophages. The mRNA expressions of some important cytokines, including tumor necrosis factor-α, interferon-γ, interleukin （IL）-1 and IL- 10, were detectable in the macrophages cocultured with mycobacteria and PBMCs, and the secretion of these cytokines was confirmed by ELISA in the supematant of these cells. Conclusions An in vitro model of myeobacterial granuloma is basically established, which may facilitate the investigation into the formation of granuloma caused by and immune response to mycobacterial infection.

关 键 词：肉芽肿 分枝杆菌属 免疫 

分 类 号：R378.91[医药卫生—病原生物学]