Abstract: Tian Weiwei, Zhang Xiaodong, Wang Qiuling, Tang Meiyu, Shen Jianping, Wang Hongsheng, Wang Qianqiu. Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China Corresponding authors: Wang Hongsheng, Email: whs33@vip.sina.com; Wang Qianqiu, Email: wangqq@ncstdlc.org 【Abstract】 Objective To establish an in vitro model of mycobacterial granuloma. Methods Mononuclear cells 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 Mycobacterium marinum, Mycobacterium tuberculosis, Bacillus Calmette-Guérin, and Mycobacterium 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 mycobacterial granuloma. The macrophages cocultured 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 macrophages aggregated to form granuloma-like clumps, and some cells fused to form multinuclear 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）-1β 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 supernatant of these cells. Conclusions An in vitro model of mycobacterial granuloma is basically established, which may facilitate the investigation into the formation of granuloma caused by and immune response to mycobacterial infection.

Key words: Granuloma, Mycobacterium, Immunity