【作者】 王婷；

【导师】 蒋豪；

【作者基本信息】 复旦大学 ， 麻醉学， 2003， 博士

【摘要】 【目的】研究异氟醚对大鼠肺缺血再灌注损伤下肺组织NF-κB结合活性及粘附分子CD18、ICAM-1和细胞因子诱导的中性粒细胞趋化物（CINC）基因表达的影响，结合肺微血管通透性、肺组织氧化损伤情况、肺内PMN聚集程度和肺组织形态学的改变，从组织到分子水平初步探讨异氟醚对肺缺血再灌注损伤是否具有保护作用及其可能的作用机制，为临床麻醉实施中麻醉药物的选择提供实验和理论的依据。【方法】建立在体大鼠肺缺血再灌注模型。120只SD雄性大鼠，随机分成四组，每组30只。手术对照组（C组）：开胸游离左肺门后，未予阻断左肺门行缺血再灌注处理；缺血再灌注组（IR组）：开胸游离左肺门后，阻断左肺门45分，而后松开血管夹形成再灌注；异氟醚-缺血再灌注组（ISO-IR组）：1MAC异氟醚持续吸入30分后，开胸游离左肺门，阻断左肺门45分后松开血管夹形成再灌注；异氟醚组（ISO-C组）：1MAC异氟醚持续吸入30分后，开胸游离左肺门但不予以缺血再灌注处理。各组分别在缺血45分、再灌注30分、60分、120分（或对照组相应时间点）放血处死6只大鼠，留取血和肺组织标本；在各组再灌注120分（或对照组相应时间点）时，另处死6只大鼠行支气管肺泡灌洗并采集血标本。持续监测MAP，并于各时间点分别进行动脉血气分析、肺组织湿干比（W/D）、MDA、SOD值及MPO活性测定和肺组织病理学检查，于再灌注120分（或对照组相应时间点）行左侧肺灌洗取支气管肺泡灌洗液（BALF）进行白细胞计数、沉渣白细胞分类和BALF中的蛋白含量测定；此外，在各组不同时间点分别运用流式细胞术检测PMN膜表面CD18的表达，RT-PCR技术检测肺组织ICAM-1 mRNA和CINC mRNA的表达及运用EMSA方法测定肺组织中NF-κB结合活性的变化。【结果】（1）在基础状态及单纯缺血时，各组间及组内PaO2之间并无显著性差异。再灌注后，IR组和ISO-IR组的PaO2均较基础状态及单纯缺血时明显降低（P<0.05），并且与C组相比亦明显下降（P<0.05）；但异氟醚的应用并未使再灌注后PaO2的下降得到明显改善，然随再灌注时间的延长，ISO-IR组PaO2降低的程度似较IR组有逐渐缩小的趋势。（2）单纯缺血时，各组肺组织W/D值之间并无显著差异。再灌注后，IR组和ISO-IR组的W/D值逐渐升高，且显著高于C组（P<0.05）；但再灌注60min后，ISO-IR组W/D值较IR组有所下降且差异具有显著性意义（P<0.05）。ISO-C组肺组织W/D值在实验观察中变化不明显，且较C组亦无明显改变。（3）各组BALF的回收率均在75%以上，而IR组和ISO-IR组回收的BALF多呈淡红色。各组间BALF中的细胞总数变化无显著性差异，但中性粒细胞（PMN）所占的百分比IR组较C组明显升高（P<0.05），而ISO-IR组的升高并不显著且较IR组明显降低。IR组和ISO-IR组BALF中TP的含量均较C组明显升高<WP=5>（P<0.05），但ISO-IR组又低于IR组（P<0.05）；ISO-C组与C组相比并无明显差异。（4）C组和ISO-C组肺组织MDA含量和SOD的活性在观察过程中均无明显变化且两组间无显著差异。再灌注后IR组肺组织MDA含量较C组同时点均明显增加，而SOD活性明显下降（P<0.05）。ISO-IR组再灌注后各时点肺组织MDA含量虽较C组明显增加（P<0.05），但再灌注60min后的MDA值较IR组却有所改善（P<0.05）；ISO-IR组的SOD活性于再灌注后较C组同时点也有明显的降低，但与IR组相比并无显著的差异。（5）C组和ISO-C组肺组织的MPO活性在观察过程中虽有逐渐增加的趋势，但变化不明显且两组间无显著差异；而IR组和ISO-IR组随再灌注时间的延长MPO活性逐渐升高，再灌注60min后MPO的活性显著高于再灌注前且较C组亦明显升高（P<0.05）；但ISO-IR组再灌注60min后的MPO活性要低于IR组（P<0.05）。（6）C组和ISO-C组肺组织ICAM-1 mRNA在整个实验观察中均呈低表达。再灌注60min后，IR组和ISO-IR组ICAM-1 mRNA的表达明显高于C组且较单纯缺血时亦明显升高（P<0.05），但ISO-IR组ICAM-1 mRNA的表达于再灌注60min后较IR组有所下降且差异具有显著性意义（P<0.05）。（7）单纯缺血期及再灌注30min时，四组之间CINC mRNA的表达并无显著差异。但在C组和ISO-C组也观察到，长时间持续通气灌注下（C165）肺组织CINC mRNA的表达较C45时有明显升高（P<0.05）。再灌注60min后，IR组和ISO-IR组肺组织CINC mRNA表达较C组和再灌注前均明显上调（P<0.05）；但ISO-IR组CINC mRNA的表达与IR组相比可见有显著意义的降低（P<0.05）。（8）C组和ISO-C组在观察中各时点CD18的表达均无显著性差异。IR组再灌注30min后，CD18的表达较单纯缺血时明显升高且随时间的延长更为显著（P<0.05），而ISO-IR组再灌注后CD18的表达虽较C组明显升高（P<0.05），但与IR组相应时点比较却见有显著性意义的改善（P<0.05）。（9）IR组和ISO-IR组肺组织ICAM-1 mRNA、CINC mRNA和PMN表面CD18的表达与MPO的活性之间均具有明显的相关性（P<0.05）。（10）C组和ISO-C组NF-κB活性在实验各观察点之间的变化并不明显且两组之间亦无显著差异；IR组和ISO-IR组肺组织NF-κB的活性于再灌注30min后均较C组明显增加（P<0.05），但与同时点IR组相比，异氟醚的吸入使ISO-IR组NF-κB的结合活性受到一定程度的抑制（P<0.05）。（11）IR组ICAM-1 mRNA、CI更多还原

【Abstract】 Objective In this study, we established a rat model of lung ischemia-reperfusion injury to observe the changes of pulmonary function, vascular permeability, the content of MDA and SOD, MPO activity, pulmonary histology, the gene expressions of intercellular adhesion molecule-1 (ICAM-1) and cytokine-induced neutrophil chemoattractant (CINC), the expression of CD18 and the DNA binding activity of nuclear factor-κB (NF-κB) in rat lung in order to explore the effects of isoflurane administration before ischemia on ischemia-reperfusion injury in rat lung and offer the experimental and theoretical evidence for the choice of anesthetics in clinical practice. Methods The rat ischemia model with occlusion of left pulmonary hilum for 45min was used in this experiment. One hundred and twenty male Sprague-Dawley rats were randomly divided into four groups. Group C (n=30): continuous perfusion without ischemia; group IR (n=30): interruption of perfusion and ventilation for 45min, followed by reperfusion; group ISO-IR (n=30): 1MAC isoflurane inhalation for 30min before ischemia, followed by ischemia-reperfusion; group ISO-C (n=30): 1MAC isoflurane inhalation for 30min, followed by continuous perfusion. The changes of arterial blood gas analysis, the lung wet-to-dry weight ratio (W/D), malondialdehyde (MDA), superoxide dismutase (SOD) and myeloperoxidase (MPO) of lung were studied at 45 min after pulmonary ischemia and 30min, 60min, 120min of reperfusion. The gene expressions of ICAM-1 and CINC were detected by reverse-transcription polymerase chain reaction (RT-PCR) , the expression of integrin subunit CD18 was determined by the method of flow cytometry and the DNA binding activity of NF-κB in rat lung was measured by electrophoretic mobility shift assay (EMSA ) at the same time above-mentioned. The cell counts, percentage of polymorphonuclear neutrophil (PMN) and total protein (TP) content in left bronchoalveolar lavage fluid (BALF) was performed at 120min of reperfusion. MAP was continuously monitored. Results (1) The change of PaO2: In IR and ISO-IR groups, PaO2 decreased gradually and was obviously lower than that in C and ISO-C groups during reperfusion (P<0.05).There was no significant difference in PaO2 between IR and ISO-IR groups, but there was a tendency of the improvement in PaO2 of ISO-IR group during reperfusion compared with IR group. (2) The lung<WP=8>W/D: At 45min after ischemia, there was no significant difference in the lung W/D among groups. During reperfusion the lung W/D in IR and ISO-IR groups increased progressively and was evidently higher than that in C and ISO-C groups (P<0.05)；but compared with IR group, preadministration of isoflurane could inhibit the increase in the lung W/D after 60min of reperfusion (P<0.05). The lung W/D in ISO-C group was no obvious change compared with C group during the experiment. (3) The cell counts, percentage of PMN and TP content in BALF: There was no marked difference in the cell counts among groups, but the percentage of PMN in IR group was obviously increased compared with the other groups at 120min of reperfusion (P<0.05). IR significantly increased the content of TP in BALF of IR and ISO-IR groups, but the degree of increase in TP content of ISO-IR group was decreased markedly compared with IR group (P<0.05). (4) The lung contents of MDA and SOD: Little change in lung contents of MDA and SOD was measured in C and ISO-C groups during the perfusion and there was no marked difference between the two groups, too. In IR and ISO-IR groups, MDA increased and SOD decreased significantly compared with C group during reperfusion (P<0.05), but the inhalation of isoflurane could attenuate the increase in MDA after 60min of the reperfusion (P<0.05) and not inhibit the decrease in SOD. (5) Lung MPO activity: The marked change of MPO activity was not observed in C and ISO-C groups during the perfusion. After the reperfusion, lung MPO activity in IR and ISO-IR groups increased gradually, especially after 60min of reperfusion and was higher than that in C group (P<0.0更多还原