OBJECTIVETo establish a model of hypoxic postconditioning of dermal microvascular endothelial cells.

METHODSDuring reoxygenation after hypoxia, cells received three times of hypoxic/ reoxygenation alternate treatment for a certain time. The cells were seeded on 6-well plates, with one plate for one group. They were divided into 6 groups as group 1 ( Control), group 2 (8h hypoxia + 24h reoxygenation), group 3 (8h hypoxia + 2 min x 3 times post-hypoxia treatment) , group 4 (8h hypoxia + 5 min x 3 times post-hypoxia treatment), group 5 (8h hypoxia + 10 min x 3 times post-hypoxia treatment), 6 group (8h hypoxia + 20 min x 3 times post-hypoxia treatment). Each group underwent 8 h hypoxia + 24 h hypoxia Buffer and reoxygenation. Lactate dehydrogenase (LDH) was detected during the process. Apoptosis rate was calculated by staining Tunel method. Bcl-2, Bax and activated caspase-3 protein were detected by Western Blot.

RESULTSIn the continuous hypoxia process, the LDH was (1563 ± 83.35) IU/L at 8h and (582.85 ± 58.25 ) IU/L at 0h, showing a statistical difference (P = 0.0001). Western blotting results showed that the expression of Bax/Bcl-2 in group 2 was 0.38 ± 0.02, showing a significant difference when compared with that in group 3 (0.23 ± 0.01) and group 4 (0.22 ± 0.02) (P = 0.012, P = 0.005), while not when compared with that in group 5 (0.33 ± 0.02) and 6 groups (0.34 ± 0.01) P > 0.05). The ratio of Activated caspase-3/caspase-3 in group 2 (6.30 ± 1.50) was significantly higher than that in group 3 (2.17 ± 0.26) and group 4 (2.63 ± 0.31) (P = 0.008, P = 0.019); while not in group 5 (4.36 ± 0.29) and group 6 (4.97 ± 0.51) (P > 0.05).

CONCLUSIONSThe model of hypoxic postconditioning of human dermal microvascular endothelial cells is successfully established.