中文摘要：

Alterations of the autophagy-lysosomal pathway(ALP) and autophagy have been involved in lung ischemia-reperfusion(I/R) injury. However, dynamic imaging of ALP function under lung I/R injury particularly is not fully understood. Here we depicted the live-cell fluorescence imaging of autophagosome to monitor ALP activation and autophagy function. The p As Red2-N1-LC3 vectors were transfected into CRL-2192 NR8383(an alveolar macrophage cell line) and CCL149(an alveolar epithelial cell line) successfully. 0-h, 2-h, 4-h, and 6-h hypoxia/0-h, 2-h, 4-h, and 6-h reoxygenation were then induced with an ALP inhibitor(3-MA) or activator(rapamycin) in the culture of transfected cells separately. ALP activation was conformed by up-regulating AMPK and beclin1 expression. Apoptosis was not obvious in 2-h hypoxia/2-h reoxygenation. p As Red2-N1-LC3 CCL149 and p As Red2-N1-LC3 NR8383 cells revealed gradually enhanced As Red2 from 2-h to 6-h hypoxia/reoxygenation. As Red2 varied sensitively to 3-MA and rapamycin interventions during 2-h hypoxia/reoxygenation. Our data provides a simple method of autophagosome imaging to monitor ALP activation and autophagy function in lung I/R injury.

英文摘要：

Alterations of the autophagy-lysosomal pathway（ALP） and autophagy have been involved in lung ischemia-reperfusion（I/R） injury. However, dynamic imaging of ALP function under lung I/R injury particularly is not fully understood. Here we depicted the live-cell fluorescence imaging of autophagosome to monitor ALP activation and autophagy function. The p As Red2-N1-LC3 vectors were transfected into CRL-2192 NR8383（an alveolar macrophage cell line） and CCL149（an alveolar epithelial cell line） successfully. 0-h, 2-h, 4-h, and 6-h hypoxia/0-h, 2-h, 4-h, and 6-h reoxygenation were then induced with an ALP inhibitor（3-MA） or activator（rapamycin） in the culture of transfected cells separately. ALP activation was conformed by up-regulating AMPK and beclin1 expression. Apoptosis was not obvious in 2-h hypoxia/2-h reoxygenation. p As Red2-N1-LC3 CCL149 and p As Red2-N1-LC3 NR8383 cells revealed gradually enhanced As Red2 from 2-h to 6-h hypoxia/reoxygenation. As Red2 varied sensitively to 3-MA and rapamycin interventions during 2-h hypoxia/reoxygenation. Our data provides a simple method of autophagosome imaging to monitor ALP activation and autophagy function in lung I/R injury.