VILI小鼠血清内皮细胞微粒变化

Changes in circulating microparticles in mice with ventilator-induced lung injury

目的 观察呼吸机相关性肺损伤（VILI）小鼠血清内皮细胞微粒（EMPs）的变化,并探讨其在VILI中的意义.方法 将48只SPF级雄性野生C57BL/6J小鼠按随机数字表法分为两组,每组24只：机械通气（MV）组小鼠于气管插管后进行30 mL/kg大潮气量（VT）MV 4 h;自主呼吸组小鼠气管插管后进行自主呼吸4 h.两组分别取12只小鼠心尖血,采用酶联免疫吸附试验（ELISA）测定血清白细胞介素（IL-1β、IL-6）和肿瘤坏死因子-α（TNF-α）水平,采用流式细胞仪检测血清EMPs水平;EMPs与IL-1β、IL-6、TNF-α的关系采用线性回归分析.另取12只小鼠肺组织,测定肺湿/干重（W/D）比值;苏木素-伊红（HE）染色后,光镜下观察肺组织形态学改变;醋酸铀-柠檬酸铅双染后,透射电镜下观察肺组织超微结构改变.结果 与自主呼吸组比较,MV组小鼠肺W/D比值明显升高（5.47±0.14比4.34±0.11）,血清IL-1β、IL-6、TNF-α及EMPs水平亦明显升高〔IL-1β（ng/L）：42.4±4.4比7.7±3.6,IL-6（ng/L）：1239.5±66.3比21.7±4.6,TNF-α（ng/L）：237.6±25.8比37.1±19.1,EMPs（个/μL）：28.6±1.8比5.9±1.8,均P〈0.01〕.相关分析显示,血清EMPs与IL-1β、IL-6、TNF-α均呈线性正相关（r值分别为0.968、0.932、0.945,均P=0.000）;拟合线性回归分析显示, EMPs每增加1个/μL,IL-1β升高2.4 ng/L〔95%可信区间（95%CI）=1.9~2.8,P〈0.001〕,IL-6升高34.5 ng/L （95%CI=25.1~44.0,P〈0.001）,TNF-α升高13.6 ng/L（95%CI=10.3~16.9,P〈0.001）.光镜下显示：自主呼吸组小鼠肺泡结构完整;而MV组肺泡萎缩或消失,肺实变,部分肺泡融合,肺泡隔增粗.电镜下显示：自主呼吸组小鼠肺泡壁完整、无渗出;而MV组肺泡壁水肿、破裂,Ⅱ型肺泡上皮细胞空泡化.结论 给予小鼠30 mL/kg VT通气4 h可导致VILI伴EMPs增加,提示EMPs水平升高与VILI的发生密切相关,可能成为VILI的标志物.

 Objective To investigate the changes in serum level of microparticles （EMPs） in mice with ventilator-induced lung injury （VILI）, and explore its significance in VILI. Methods Forty-eight grade SPF male C57BL/6J mice were randomly divided into two groups, with 24 mice in each group： the mice in mechanical ventilation （MV） group were given high tidal volume （VT 30 mL/kg） MV for 4 hours after tracheal intubation, and those in spontaneous breathing group were spontaneously breathed for 4 hours. The apical blood of 12 mice in each group were collected, and serum levels of interleukins （IL-1β, IL-6） and tumor necrosis factor-α （TNF-α） were determined by enzyme linked immunosorbent assay （ELISA）, and serum EMPs levels were determined by flow cytometer. The correlations between EMPs and IL-1β, IL-6, and TNF-α were analyzed by linear regression analysis. The lung tissues of other 12 mice in each group were harvested, and wet/dry weight （W/D） ratio was assessed. After hematoxylin-eosin （HE） staining, the morphological changes in lung tissue were observed under light microscope. After double staining of uranium acetate and lead citrate, the ultrastructural changes in lung tissue were observed with electron microscope. Results Compared with spontaneous breathing group, the levels of lung W/D ratio in MV group was significantly increased （5.47±0.14 vs. 4.34±0.11）, the levels of IL-1β, IL-6, TNF-α and EMPs were also significantly increased [IL-1β （ng/L）： 42.4±4.4 vs. 7.7±3.6, IL-6 （ng/L）： 1 239.5±66.3 vs. 21.7±4.6, TNF-α （ng/L）：237.6±25.8 vs. 37.1±19.1, EMPs （cells/μL）： 28.6±1.8 vs. 5.9±1.8, all P 〈 0.01]. It was shown by correlation analysis that EMPs were positively related with IL-1β, IL-6, and TNF-α （r value was 0.968, 0.932, 0.945, respectively, all P = 0.000）. It was shown by fitting linear regression analysis that when EMPs increased by 1 cell/μL, IL-1β increased by 2.4 ng/L [95% confidence interval （95%CI） = 1.9-2.8, P 〈 0.001], IL-6 increased by 34.5 ng/L （95%CI = 25.1-44.0, P 〈 0.001）, and TNF-α increased by 13.6 ng/L （95%CI = 10.3-16.9,P 〈 0.001）. It was shown by light microscope that the structure of lung tissue and alveolar of mice in spontaneous breathing group appeared normal, while the shrinks of alveolar and disappearance of alveolar architecture were found in MV group. It was shown by electron microscopy that alveolar wall edema and thickening and broken alveolar septa were found in MV group, by contrast, the structure of alveolar was normal in spontaneous breathing group. Conclusion 30 mL/kg VT ventilation for 4 hours could induce VILI with increase in EMPs, suggesting EMPs closely related to VILI, and EMPs level may be putative biomarker of VILI.