不同潮气量机械通气对大鼠肺小窝蛋白-1及其相关信号链酶表达的影响

发布时间：2018-09-13 07:36

【摘要】：目的：研究小窝蛋白-1（caveolin-1cav-1）在不同潮气量机械通气时大鼠肺组织表达的变化及其调控酶、下游信号链炎症因子、肺组织病理生理变化及物理力学变化，通过上述研究，试图阐明小窝蛋白-1及相关信号链酶对机械通气相关性肺损伤的调控，并为临床防治肺损伤提供新的理论机制和思考点。方法：成年健康雄性SD大鼠40只，体重200-340g，随机分为5组(n=8)，对照组(A组)仅切开气管保留自主呼吸；不同通气时间保护性机械通气组(B1组和B2组)，潮气量（tidal volume VT）：6ml/kg，频率（respiratory rate RR）：60次/分，吸呼比（I/E）：1：2，分别通气1h或2h；不同通气时间大潮气量机械通气组(C1组和C2组)，，潮气量（VT）：30ml/kg，频率（RR）：40次/分，吸呼比（I/E）：1：2，分别通气1h或2h。A组在气管切开即刻，其余各组在机械通气结束时处死大鼠，光镜下观察肺组织病理改变并作弥漫性肺泡损伤系统（diffuse alveolar damageDAD）评分；免疫组织化学染色法检测肺组织中cav-1、内皮型一氧化氮合酶（endothelial nitric oxide synthase eNOS）、白介素-1受体相关激酶4（interleukin-1receptor-associated kinase4IRAK4）及核转录因子-κB（nuclear factor-kappaB NF-κB）p65蛋白的表达；逆转录-聚合酶链式反应（reverse transcription-polymerase chain reaction RT-PCR）测定cav-1、eNOSmRNA的表达；比色法测定髓过氧化物酶(myeloperoxidaseMPO)活性；计算肺湿干质量比值(W/D)；酶联反应吸附实验（enzyme-linkedimmunosorbent assay ELISA）测定支气管肺泡灌洗液(bronchoalveolarlavage fluid BALF)中肿瘤坏死因子-α（tumor necrosis factor-α TNF-α）含量。各组大鼠均连续监测平均动脉压（mean arterial pressure MAP）及血气分析。A组在测定基础MAP、基础动脉血气后立即放血处死，采集标本。B1组和C1组分别在通气0h、1h测定动脉血气，通气1h后放血处死，采集标本。B2组和C2组分别在通气0h、1h、2h测定动脉血气，通气2h后放血处死，采集标本。结果：A组、B1组、B2组之间肺组织cav-1、eNOS的mRNA表达，cav-1、eNOS、IRAK4、NF-κBp65的蛋白表达，W/D比值，MPO水平和TNF-α含量差异均无统计学意义（均P＞0.05）。与A组比较，C1组、C2组cav-1、IRAK4、NF-κB蛋白及cav-1mRNA表达上调，W/D比值、MPO、TNF-α含量和病理损伤评分升高(均P0.05)。与B1组比较，C1组cav-1、IRAK4、NF-κB p65蛋白及cav-1mRNA表达上调，W/D比值、MPO、TNF-α含量和病理损伤评分升高(均P0.05)。与B2组比较，C2组cav-1、IRAK4、NF-κB p65蛋白及cav-1mRNA表达上调，W/D比值、MPO、TNF-α含量和病理损伤评分升高(均P0.05)。与A组比较，C1组、C2组eNOS蛋白及eNOSmRNA表达下调(均P0.05)。与B1组比较，C1组eNOS蛋白及eNOSmRNA表达明显减少(均P0.05)。与B2组比较，C2组eNOS蛋白及eNOSmRNA表达明显减少(均P0.05)。与C1组比较，C2组eNOS蛋白及eNOSmRNA表达明显减少(均P0.05)。机械通气前，5组大鼠动脉血气和MAP无统计学差异（均P＞0.05）。机械通气开始后，与相应B1、B2组比较，C1、C2组动脉血气PH值升高、PaO2、PaCO2、MAP均逐渐下降，差异有统计学意义(均P0.05)。结论：小窝蛋白-1及其相关信号链酶在机械通气中表达的上调表明，小窝蛋白-1参与了机械通气相关性肺损伤的发生。
[Abstract]:AIM: To study the changes of caveolin-1 (cav-1) expression and its regulatory enzymes, downstream signal chain inflammatory factors, pathophysiology and physical and mechanical changes in lung tissues of rats undergoing mechanical ventilation with different tidal volumes. Methods: Forty adult healthy male SD rats weighing 200-340g were randomly divided into 5 groups (n=8), control group (group A) only incised trachea to retain spontaneous respiration, protective ventilation group (group B1 and B2) with different ventilation time and tidal volume VT (6ml/kg). Respiratory rate RR: 60 times per minute, inspiratory-expiratory ratio (I/E): 1:2, ventilated for 1 hour or 2 hours, ventilated with high tidal volume (C1 and C2) at different ventilation time, tidal volume (VT): 30 ml/kg, frequency (RR): 40 times per minute, inspiratory-expiratory ratio (I/E): 1:2, ventilated for 1 hour or 2 hours, respectively. Group A immediately after tracheotomy, the rest of the groups at the end of mechanical ventilation. The pathological changes of lung tissue were observed under light microscope and diffuse alveolar damage (DAD) was scored; cav-1, endothelial nitric oxide synthase (NOS) and interleukin-1 receptor-associated kinase 4 (IL-1 receptor-associate) were detected by immunohistochemical staining. The expression of edkinase 4IRAK4 and nuclear factor kappa B NF-kappa B p65 protein, the expression of Cav-1 and eNOSmRNA, the activity of myeloperoxidase (MPO) and the ratio of wet to dry lung mass (W/W) were measured by reverse transcription-polymerase chain reaction (RT-PCR), the activity of myeloperoxidase (MPO) by colorimetry, and the expression of Cav-1 and eNOSmRNA by reverse transcription-polymerase chain reaction (RT-PCR). The levels of tumor necrosis factor - alpha TNF - alpha in bronchoalveolar lavage fluid BALF were measured by enzyme - linked dimmunosorbent assay (ELISA). Mean arterial pressure MAP and blood gas analysis were continuously monitored in all groups. Basal MAP, basal arterial blood gas immediately after bleeding, collected specimens. B1 group and C1 group were ventilated 0 h, 1 h arterial blood gas, ventilated 1 h after bleeding, collected specimens. B2 group and C2 group were ventilated 0 h, 1 h, 2 h arterial blood gas, ventilated 2 h after bleeding and killed, collected specimens. There was no significant difference in NA expression, cav-1, eNOS, IRAK4, NF-kappa Bp65 protein expression, W/D ratio, MPO level and TNF-a content (all P > 0.05). Compared with group A, cav-1, IRAK4, NF-kappa B protein and Cav-1 mRNA expression were up-regulated in group C1 and C2, W/D ratio, MPO, TNF-a content and pathological injury score were increased (all P 0.05). Compared with B2 group, cav-1, IRAK4, NF-kappa B p65 and Cav-1 mRNA were up-regulated, W/D ratio, MPO, TNF-a and pathological injury score were up-regulated, and the expression of eNOS protein and eNOSmRNA were down-regulated in C2 group (all P 0.05). Compared with group B1, the expression of eNOS protein and eNOSmRNA in group C1 decreased significantly (all P 0.05). Compared with group B2, the expression of eNOS protein and eNOSmRNA in group C2 decreased significantly (all P 0.05). Compared with group C1, the expression of eNOS protein and eNOSmRNA in group C2 decreased significantly (all P 0.05). Before mechanical ventilation, there was no significant difference in arterial blood gas and MAP between the five groups (all P > 0.05). After mechanical ventilation, compared with the corresponding B1 and B2 groups, the PH value of arterial blood gas in C1 and C2 groups increased, while the PaO2, PaCO2 and MAP decreased gradually (all P 0.05).
【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R563.8

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