The role of platelet in mouse pulmonary microvascular endothelial cell injury induced by lipopolysaccharide-activated neutrophil in vitro / 中华麻醉学杂志

ABSTRACT

ObjectiveTo investigate the role of platelet in mouse pulmonary microvascular endothelial cell (PMVEC) injury caused by lipopolysaccharide( LPS)-activated neutrophil.MethodsPMVECs were obtained from pathogen-free C3H/HeN mice of both sexes aged 6-8 weeks weighing 18-25 g according to the method described by Lim YC et al.Platelets and neutrophils were isolated from mouse blood by twice centrifugation and denaity gradient centrifugation respectively.PMVECs were seeded into twelve- or six-well plates ( 1 or 2 ml/well) after 2-5 passages and were randomly divided into 4 groups (n =31 each) group LPS; group platelets (group P);group neutrophils (group N) and group platelets + neutrophils (group PN).Each well contained about 5 × 107/ml platelets and/or 5 × 105/ml neutrophils respectively.PMVECs were incubated with LPS1 μg/ml at 37 ℃ in a 5％ CO2 humidified atmosphere for 1,6,12,18 and 24 h respectively in all 4 groups.The cells were examined with phase contrast microscope for morphological changes and survival condition.Viability rate,apoptotic rate and activation rate of PMVECs were detected by flow cytometry at each time points.ResultsThere was no significant difference in morphology and number of endothelial cells (ECs) among the 4 groups,while the number of activated ECs was significantly increased but the number of living cells decreased in group PN compared with group LPS.The activation rate of ECs was significantly higher after being incubated with LPS for 6-12 h in groups P and N than in group LPS.The viability rate was significantly lower,while the apoptotic rate and activation rate were significantly higher after ECs were incubated with LPS in group PN than in groups LPS,P and N.ConclusionPlatelets play a decisive role in mouse PMVEC injury induced by LPS activated neutrophils.