3D结核球模型的构建及特性验证:基于人髓系THP-1细胞与卡介苗

Characterization of a 3-dimensional tuberculosis spheroid model constructed using human monocytic THP-1 cells and Bacillus Calmette-Guerin

目的为研究体内结核性肉芽肿的形成及特性提供体外实验模型。方法将人髓系白血病单核细胞(THP-1)和结核分枝杆菌的模式菌卡介苗(BCG)按一定比例混合加入3D细胞培养平板,再加入佛波脂(PMA)共培养3d。采用倒置荧光显微镜每24h观察球体的生长情况,在共聚焦激光扫描显微镜下分别观察结核球内BCG的分布、球体内细胞的存活率和单核细胞向巨噬细胞转变情况,并观察荧光标记的纳米粒在细胞球和结核球渗透情况。通过透射电镜观察3D结核球形成后内部细菌和细胞的微观结构。利用Image-iT^(TM)红色缺氧探针,H_(2)O_(2)检测试剂盒和防水笔形PH计检测受BCG感染的3D结核球和未受BCG感染的细胞球微环境的差异。通过平板菌落计数检测3D结核球能否评估利福平或左氧氟沙星抑菌能力。结果倒置荧光显微镜显示细胞-细菌悬浮液随时间延长缓慢堆积形成稳定的3D结核球,直径50~200μm。激光共聚焦显微镜可见细胞黏附紧密形成球样结构,大量细菌主要分布于细胞中心,球体内部出现坏死细胞,3D结核球内大量单核细胞成功分化为巨噬细胞。与单纯细胞球比较药物难以渗透入3D结核球中心。透射电镜结果显示结核球内有细胞坏死且坏死程度不一,大量BCG被吞噬于巨噬细胞内部。微环境检测结果显示与未受感染的细胞球比BCG感染的结核球更加缺氧,H_(2)O_(2)含量更高,PH接近中性。平板菌落计数结果显示一线抗结核药物利福平有更强抑菌效果,结核球有评估抗结核药物强弱作用。结论成功建立了体外3D结核球模型并对其特性进行了验证,有望为研究结核性肉芽肿相关研究奠定基础。

Objective To establish a 3-dimensional tuberculosis spheroid model for studying the formation and characteristics of tuberculous granuloma in vivo.Methods Human myeloid leukemia mononuclear THP-1 cells and Bacillus Calmette-Guerin(BCG)were mixed in a 3D cell culture plate and co-cultured in the presence of PMA for 3 days.The growth of the spheroid was examined every 24 h,and the distribution of bacteria,cell survival rate,transformation of the monocytes into macrophages,and penetration of fluorescently labeled nanoparticles into the cell spheroids and tuberculosis spheroids were observed using confocal laser scanning microscopy.The BCG and cell architecture within the 3D tuberculosis spheroid was observed using transmission electron microscopy.Image-iT™red hypoxia probe,H_(2)O_(2) test kit,and a waterproof pen PH meter were used to detect the differences in the microenvironment between BCG-infected and non-infected 3D tuberculous spheroids.The utility of this 3D tuberculous spheroids for assessing antibiotic effects of rifampicin and levofloxacin was evaluated by plate colony counting.Results In the cell-bacterial suspensions,stable 3-D tuberculous spheroids(50-200μm)occurred slowly,in which the cells adhered tightly with numerous bacteria in the center,and necrotic cells and monocyte-derived macrophages were seen within the spheroids.Drug penetration was difficult in the 3D tuberculous spheroids as compared with the non-infected cell spheroids.Transmission electron microscopy revealed the presence of cell necrosis and a large number of BCG in the macrophages in the tuberculous spheroids.The tuberculosis spheroid had a more hypoxic microenvironment than the non-infected cell spheroids with higher H_(2)O_(2) content and nearly a neutral PH.The tuberculous spheroid model was capable of evaluating the efficacy of anti-tuberculosis drugs,and among them rifampicin showed a stronger antibacterial effect.Conclusion The 3-D tuberculous spheroid model established in this study provides a useful platform for studies of tuberculous granuloma.