诱导中性粒细胞凋亡减轻体外循环后肺损伤的体内外实验研究

In vitro and vivo experimental study on ameliorating lung damage after cardiopulmonary bypass by acceleration neutruphils apoptosis

目的观察诱导中性粒细胞(PMN)凋亡对体外循环(CPB)后肺脏损伤的保护作用。方法在体外实验中,Percoll细胞分离液梯度密度离心得PMN,培养48 h,实验组加入不同浓度的克拉霉素 (5、10、20μg/ml)。台盼蓝染色,镜下观察细胞存活率。流式细胞仪检测细胞凋亡率。免疫组化法观察 PMN凋亡相关基因蛋白Fas和bcl-2的表达情况。在体内实验中,将12只绵羊随机分为低分子右旋糖酐肺动脉灌注组(对照组)和低分子右旋糖酐+克拉霉素肺动脉灌注组(实验组)。建立CPB后经肺动脉灌注肺保护液,CPB 90min后撤离CPB。观察呼吸功能,检测细胞因子浓度,并观察肺组织形态学改变及肺内PMN凋亡情况。结果克拉霉素明显缩短PMN的生存期。流式细胞仪检测显示,实验组24 h凋亡率 1、5、10和20μg/ml浓度组分别为(33．7±4．9)％、(48．0±4．9)％、(52．0±5．4)％和(53．0±7．1)％,明显高于对照组的(31．5±3．5)％(P<0．01);免疫组化染色显示实验组Fas的表达较对照组高,而bcl-2的表达低于对照组(P<0．01);体内实验结果显示,实验组肺血管阻力[(10．22±1．44)kPa·s·L-1]低于对照组 [(20．26±4．71)kPa·s·L-1,P<0．01],而动脉血氧指数[(188±48)mm Hg]较对照组[(123±62)mm Hg, P<0．05]高。实验组支气管肺泡灌洗液内细胞因子白细胞介素-8和肿瘤细胞因子均低于对照组(P< 0．05)。形态学观察表明实验组肺损伤较对照组轻。对照组肺内中性白细胞凋亡率为29％,实验组凋亡率达73％(P<0．01)。结论克拉霉素能诱导PMN凋亡,减轻CPB所造成的肺脏损伤。

Objective To observe the protective effect of acceleration neutruphils apoptosis on lung injury after cardiopulmonary bypass（CPB）. Methods The neutmphils were separated using discontinuous PercoU gradients in vitro and cultured for 48 hours with or without clacithromycin. Neutmphil survival was determined by typan blue dye exclusion method. Apoptotic neutmphils were detected with flow cytometry. Immune histochemistry was used to examine the expression of protein of Fas and bcl-2 which interrelated with neutmphil apoptosis. In vivo study twelve adult sheeps were randomly divided into two groups. Pulmonary artery was clamped after CPB was established and the lungs were peffused with 4℃ low molecular weight dextran solution in control group （n = 6） and 4℃ low molecular weight dextran solution with clarithromycin in protective group （n = 6）. The bypass was withdrawn after 90 minutes. Respiratory function were recorded during CPB and cytokines in the lung were measured. Lung biopsies and the apoptosis of the neutrophil were also performed after operation. Results Clarithromycin significantly shortened nentrophil survival in a dose-dependent fashion. The result of flow cytometry showed that the apoptosis rates at 24h of clarithromycin group were （ 33.7 ± 4.9） %, （48.0 ± 4.9） %, （52.0 ± 5.4） % and （53.0 ± 7.1 ）% at 1, 5, 10 and 20 μg/ml of clarithromycin respectively while the rate in control group was （31.5 ± 3.5）% （P 〈 0.01 ）. The expression of protein of Fas treated with clacithromycin were upregulated compared with control group while the expression of bcl-2 were downregulated in control group（P 〈 0.01 ）. Ptdmonary vascular resistance[ （10.22 ± 1.44） kPa·s· L^-1 ] were higher in control group compared with protective group[ （20.26 ± 4.71 ） kPa·s·L^-1, P 〈 0.01 ] while the oxygen index were lower in control group （P 〈 0.05 ）. The cytokine interhlkin-8 and tumor necrosis factor in bronchoalveolar lavage fluid were also signifcanfly higher in control group compared to protective group （P 〈 0.05）. Histologic analyses show intraalveolar hemorrhage and neutrophil accumulation in control groups. The proportion of apoptesis of accumulated nentmphils in the lung waS lower in control group than in protective group and the proportion values were significantly different between groups（ P 〈 0.01 ）. Conclusion Clarithromycin can induce the apoptosis of neutruphils and ameliorate lung damage after cardiopulmonary bypass.