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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。
Objective To investigate the role of ESAT6 in the induction of immune escape (ESAT6) -mediated immunodeficiency (ESAT6) -mediated esophageal cancer cell death (ESAT6) Provide experimental basis. Methods The growth of BCG in mouse RAW264.7 macrophages transfected with control plasmid pCMV-HA, recombinant plasmid pCMV-HA-ESAT6 and Earle’s Balanced Salt Solution (EBSS) were observed. Western blot was used to detect the expression of pCMV- After RAW264.7 cells were treated with HA-ESAT6 for 0, 8, 12, 24 and 32 h, the microtubule-associated protein 1 light chain 3 (LC3) protein level was detected. The levels of LC3 were detected by Western blot under the combined treatment of pCMV-HA, pCMV-HA-ESAT6, chloroquine (CQ) and CQ and pCMV-HA-ESAT6 transfection. Lyso Tracker Red Quantity, observe the situation of LuoGen medium BCG. Results The growth of BCG in pCMV-HA-ESAT6 transfected RAW264.7 cells was vigorous, while the growth of EBSS-treated group was inhibited. At 0, 8, 12, 24 and 32 h, the expression of pCMV-HA-ESAT6 in RAW264.7 Compared with the control group, lysosomal LysoTracker Red staining showed that lysosomes of pCMV-HA-ESAT6, CQ and CQ and pCMV-HA-ESAT6 transfection groups But there was no significant difference between the latter three groups. The growth of BCG in pCMV-HA-ESAT6, CQ and CQ and pCMV-HA-ESAT6 transfection groups was vigorous. Conclusion ESAT6 can inhibit the autophagy in RAW264.7 cells and promote the growth of BCG infected in RAW264.7 cells.