肺上皮细胞自噬体在结核分枝杆菌感染中保护作用的分子机制

目的：构建Atg5．真核表达载体并瞬时转染肺上皮细胞细胞株，探讨自噬在结核分枝杆菌感染上皮细胞中保护作用的分子机制。方法：设计针对Atg5的RNAi序列，化学合成后经过变性，退火连接到pSilencerTM3．1-H1hygro真核表达载体，经测序验证其正确性。脂质体法瞬时转染真核细胞A549，免疫印迹法检测瞬时转染的效果。用结核分枝杆菌分别感染正常和自噬表达低下的A549细胞．通过检测LDH来观察细胞的坏死情况。结果：成功的构建了pSilencerTM3．1-H1hygro真核表达载体并建瞬时转染了A549细胞株，成功抑制了细胞的自噬功能。Atg5-细胞对结核杆菌的抵抗能力下降。结论：在自噬表达低下的细胞中，细胞对结核分枝杆菌的抵抗能力有明显下降。在结核分枝杆菌感染上皮细胞的过程中，自噬是一种保护机制。

The Effect of Autophagy on Mycobacterium Tuberculosis Infections of Lung Epithelial Cells and its Mechanism

Objective： To construct the eukaryotic plasmid of Atg5 and transfect A549 cells, and to investigate the role of autophagy on Mycobacterium Tuberculosis infections of lung epithelial cells and its mechanism. Methods： The RNAi primer of Atg5 was designed, and the Oligos of 64 base pairs for hairpin RNA targeting Atg5 were synthesized. Eukaryotic victor pSilencerTM 3. 1-H1 hygro was connected and DNA sequencing. The recombinant vector was transfected into A549 cells by lipofectamineTM 2000. Transient transfected A549 cell line was established and identified by Western blot. Results： The eukaryotic expression vector pSilencerTM 3.1-H1 hygro was constructed, and transient transfected A549 cell line was established. The protein was inhibited successfully. A549 cells and transient transfected A549 cell were infected with Mycobacterium Tuberculosis, and the LDH were detected. Conclusions： The capacity of A549 to resistance of Mycobacterium Tuberculosis was declined significantly in the cells with low autophagy. Autophagy is a defense mechanism in infected lung epithelial cells.