中文摘要：

目的 6 kD早期分泌型抗原靶点（ESAT6）能够促进卡介苗（BCG）的增殖,巨噬细胞的自噬功能能有效杀伤BCG,研究ESAT6与自噬的相互作用,为揭示ESAT6介导的免疫逃逸提供实验依据。方法分别观察对照质粒pCMV-HA和重组质粒pCMV-HA-ESAT6转染以及Earle平衡盐溶液（EBSS）处理的小鼠RAW264.7巨噬细胞中BCG的生长情况,Western blot法检测转染pCMV-HA-ESAT6的RAW264.7细胞0、8、12、24、32 h后,微管相关蛋白1轻链3（LC3）蛋白水平。转染pCMV-HA、pCMV-HA-ESAT6、氯喹（CQ）单独处理和CQ与pCMV-HA-ESAT6转染联合处理条件下,Western blot法检测LC3的水平,Lyso Tracker Red染料法计数溶酶体数量,观察罗琴培养基上BCG的情况。结果 pCMV-HA-ESAT6转染的RAW264.7细胞中BCG的生长旺盛,而EBSS处理组生长受抑制;在0、8、12、24、32 h,pCMV-HA-ESAT6转染的RAW264.7细胞中LC3Ⅰ向LC3Ⅱ转换逐渐增强;与对照组相比,溶酶体荧光探针Lyso Tracker Red染色法显示pCMV-HA-ESAT6、CQ以及CQ与pCMV-HA-ESAT6转染处理组的溶酶体较多且体积大,但后三组间均无显著性差异;pCMV-HA-ESAT6、CQ以及CQ与pCMV-HA-ESAT6转染处理组的BCG生长旺盛。结论 ESAT6抑制RAW264.7细胞内的自噬水平,并且能够促进RAW264.7细胞中感染BCG的生长。

英文摘要：

Objective To explore the interaction between 6 kD early secretory antigenic target （ESAT6） and autophagy in order to provide an experimental basis for the immune evasion mediated by ESAT6. Methods RAW264.7 cell tines （mouse macrophages） were treated with Earle＇s balanced salt solution （EBSS）, and another cells were transfected by control plasmid pCMV-HA or recombinant plasmid pCMV-HA-ESAT6. Then we observed the growth of Bacillus Calmette- Guerin （BCG） in macrophages. Western blotting was used to detect the LC3 levels in RAW204.7 cells at 0, 8, 12, 32 hours after transducted by pCMV-HA-ESAT6. In RAW204.7 cells transfected with PCMV-HA, PCMV-HA-ESAT6, and treated with chloroquine （CQ） and CQ combined with pCMV-HA-ESAT6, which were lysed and cultured in Lowenstein-Jensen culture medium for BCG counting, LC3 was detected by Western blot analysis, and the number and size of tysosomes were observed by LysoTracker Red staining. Results Compared with control plasmid pCMV-HA transfected RAW264. ？ cells, the number of BCG significantly increased in PCMV-HA-ESAT6-transfected cells, while decreased in EBSS-treated cells. PCMV- HA-ESAT6 transfection resulted in the increased transition of LC3 I to LC3 II in a time-depended manner. Compared with the controls, LysoTracker Red staining showed PCMV-HA-ESAT6, CQ and CQ plus PCMV-HA-ESAT6 transfections resulted in the increased number and size of lysosomes, and there were no differences among the three groups. Moreover, the growth potential of BCG was strong in the three transfection groups. Conclusion ESAT6 can inhibit the autophagy and promote the growth of BCG in RAW264.7 cells.