摘要
目的分析呼吸机相关性肺损伤（VILI）中生物伤的早期发病机制。方法8周龄雄性SD大鼠24只，按随机数字表法分为假手术组（S组）、常规机械通气组（L组）和大潮气量（VT）机械通气组（H组），每组8只。麻醉大鼠后均行气管切开术，S组大鼠不进行机械通气、自主呼吸空气；L组和H组大鼠其他呼吸机参数均一致，吸入氧浓度（FiO2）为0.21，L组VT 7 m/g，H组VT 28 m/g。4 h后处死大鼠，取肺组织测定/重（/）比值，苏木素-伊红（HE）染色后评估肺损伤程度，用原位末端缺刻标记法（TUNEL）染色后观察肺组织细胞凋亡情况，用蛋白质免疫印迹试验（Western Blot）测定肺组织中细胞膜半通道蛋白Pannexin-1的表达水平；提取支气管肺泡灌洗液（BALF），测定乳酸脱氢酶（LDH）、异前列醇和三磷酸腺苷（ATP）水平，并进行白细胞计数，用荧光染料Yo-pro-/化丙啶（PI）双染后观察早期细胞凋亡情况。结果S组与L组大鼠肺组织损伤情况差异无统计学意义。与S组和L组相比，H组大鼠肺组织出现明显的病理学损伤，表现为肺泡破裂、炎性细胞浸润、间质水肿、气道上皮脱落较明显，肺/比值明显增加（5.1±0.2比4.4±0.2、4.3±0.4，均P〈0.01），病理学评分明显升高〔4.00（4.00，8.00）比1.00（0，4.00）、2.00（0，4.75），均P〈0.01〕，BALF中白细胞数明显增加（×10/：2.97±0.46比1.03±0.26、0.79±0.19，均P〈0.01），LDH水平明显升高（/：148.6±38.2比34.4±13.5、78.6±13.9，均P〈0.01），且肺组织中Pannexin-1蛋白表达水平明显升高（Pannexin-/APDH：0.89±0.21比0.48±0.25、0.61±0.17，均P〈0.01），BALF中ATP水平明显升高（nmo/：456.84±148.72比19.23±13.34、113.26±57.90，均P〈0.01）；而3组间肺组织凋亡细胞数及BALF中细胞凋亡率、异前列醇水平差异无统计学意义〔肺组织细胞凋亡数（/P）：4.00（3.00，5.00）比5.00（4.00，6.00）、4.00（3.25，6.00），BALF中细胞凋亡率：（0.57±0.20）%比（0.42±0.16）%、（0.58±0.19）%，BALF中异前列醇（μ/）：3.85±0.46比3.83±0.60、3.59±0.69，均P〉0.05〕。结论VILI中生物伤早期发病机制与膜通道感受压力应激开放并释放炎性介质有关，而凋亡、脂质过氧化反应不是首要致病因素。
ObjectiveTo investigate the pathogenesis of early biotrauma in ventilator-induced lung injury （VILI）.MethodsTwenty-four 8-week-old male specific-pathogen-free Sprague-Dawley （SD） rats weighing 250-300 g were randomly divided into sham group （S group）, conventional mechanical ventilation group （L group） and high tidal volume （VT） mechanical ventilation group （H group） with 8 rats in each group. All rats received tracheostomy after anesthesia. Rats in S group received no mechanical ventilation but breathe room air spontaneously. All other parameters of the ventilator were the same in both mechanical ventilation groups, and the fraction of oxygen was set to 0.21, the rats in L group received 7 m/g VT, and those in H group received 28 m/g VT. Four hours after ventilation all rats were sacrificed and the lung tissues were harvested for we/ry （/） ratio. Pathological injury score was evaluated by hematoxylin and eosin （HE） staining. Transferase-mediated deoxyuridine triphosphate-biotin nick end labeling stain （TUNEL） was performed to count the apoptosis cell in lung epithelial. Western Blot was performed to evaluate hemi-channel protein Pannexin-1 expression in lung homogenate. Bronchoalveolar lavage fluid （BALF） was collected, and the concentration of lactate dehydrogenase （LDH）, isoprostane, adenosine triphosphate （ATP） and white cell count in BALF were measured. Yo-pro-/ropidium iodide （PI） double stain was performed to evaluate early apoptosis cell in BALF.ResultsThere was no significant difference in lung injury between S group and L group. Compared with S group and L group, rats in H group showed significant lung injury, represented as alveolar rupture, inflammatory cell infiltration, interstitial edema and airway epithelial exfoliation, and the lung / ratio was increased significantly （5.1±0.2 vs. 4.4±0.2, 4.3±0.4, both P 〈 0.01）, pathological score was significantly increased [4.00 （4.00, 8.00） vs. 1.00 （0, 4.00）, 2.00 （0, 4.75）, both P 〈 0.01], the white cell in BALF was significantly increased （×10/： 2.97±0.46 vs. 1.03±0.26, 0.79±0.19, both P 〈 0.01）, the level of LDH was significantly increased （/： 148.6±38.2 vs. 34.4±13.5, 78.6±13.9, both P 〈 0.01）, and the expression of Pannexin-1 in lung homogenate was significantly increased （Pannexin-/APDH： 0.89±0.21 vs. 0.48±0.25, 0.61±0.17, both P 〈 0.01）, the ATP concentration in BALF was also significantly increased （nmo/： 456.84±148.72 vs. 19.23±13.34, 113.26±57.90, both P 〈 0.01）. There was no significant difference in the apoptosis cell in lung tissue or the apoptosis cell rate, isoprostane level in BALF among the three groups [apoptosis cell in lung （cell/P）： 4.00 （3.00, 5.00） vs. 5.00 （4.00, 6.00）, 4.00 （3.25, 6.00）; apoptosis cell rate in BALF： （0.57±0.20）% vs. （0.42±0.16）%, （0.58±0.19）%; isoprostane in BALF （μ/）： 3.85±0.46 vs. 3.83±0.60, 3.59±0.69, all P 〉 0.05].ConclusionThe early pathogenesis of biotrauma in VILI is related to the release of inflammation mediator via membrane channel after activating by pressure stress, but not apoptosis and lipid peroxidation.
作者
贾佳
李娜
李国福
臧彬
Li Guofu;Zang Bin(Department of lntensive Care Unit,Shengjing Hospital of China Medical University,Shenyang 110004,Liaoning,China)
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2018年第9期861-866,共6页
Chinese Critical Care Medicine
基金
国家自然科学基金（81471847）
