血小板在脂多糖激活中性粒细胞致小鼠肺微血管内皮细胞损伤中的作用

The role of platelet in mouse pulmonary microvascular endothelial cell injury induced by lipopolysaccharide-activated neutrophil in vitro

目的评价血小板在脂多糖（LPS）激活中性粒细胞致小鼠肺微血管内皮细胞（PMVEC）损伤中的作用。方法C3H／HeN小鼠，6～8周龄，雌雄不拘，体重18，25g，原代培养PMVEC，采用二次离心法分离血小板，采用密度梯度离心法分离中性粒细胞。取2．5代的PMVEC，以10^5个／ml密度接种于12孔（1ml／孔）或6孔（2ml／孔）培养板，采用随机数字表法，将其随机分为4组（n=31）：LPS组、血小板组（P组）、中性粒细胞组（N组）和血小板＋中性粒细胞组（PN组）。4组均加入终浓度为1μg／ml的LPS，P组和N组分别加入血小板和中性粒细胞，PN组加入血小板和中性粒细胞，血小板和中性粒细胞的终浓度分别为5×10^7个／ml和5×10^5个／ml。分别于孵育1、6、12、18和24h时每组取1孔，相差显微镜下观察内皮细胞形态和活化情况；于孵育24h时每组取1孔，荧光显微镜下观察细胞存活情况；分别于孵育1、6、12、18和24h时取6孔，采用流式细胞术测定内皮细胞存活率、凋亡率及活化率。结果与LPs组比较，N组和P组内皮细胞形态、数量均无明显改变，而PN组活化的内皮细胞增加，活细胞数量减少；与LPS组比较，PN组LOS孵育6—24h时细胞存活率降低，细胞凋亡率升高，LPS孵育1—24h时细胞活化率升高，P组和N组LPS孵育6、12h时细胞活化率升高（P〈0．05），其余指标差异均无统计学意义（P〉0．05）；与N组或P组比较，PN组LPS孵育6—24h时细胞存活率降低，细胞凋亡率和细胞活化率升高（P〈0．05）。结论血小板在LPS激活中性粒细胞致小鼠PMVEC损伤中起决定性作用。

Objective To investigate the role of platelet in mouse pulmonary microvascular endothelial cell （PMVEC） injury caused by llpopolysacchafide（LPS）-activated neutrophil. Methods PMVECs 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 density gradient centrifugation respectively. PMVECs were seeded into twelveor 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 5×10^7/ml platelets and/or 5×10^5/ml neutrophils respectively. PMVECs were incubated with LPS 1 μg/ml at 37 ℃ in a 5% CO2 humidified atmosphere for 1, 6, 12, 18 and 24 h respectively in all 4 groups. The ceils 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. Results There was no significant differenee in morphology and number of endothelial cells （ECs） among the 4 groups, while the number of activated ECs was significantly increased but the numher of living cells decreased in group PN compared with group LPS. The activation rate of ECs was significantly higher alter being incubated with LPS for 6-12 h in groups P and N than in group LPS. The viability rzte 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. Conclusion Platelets play a decisive role in mouse PMVEC iujnry induced by LPS activated neutrophils.