【作者】 郑志坤；

【导师】 王建军； 李劲松；

【作者基本信息】 华中科技大学 ， 胸外科， 2013， 博士

【摘要】 第一部分短时长吸入一氧化氮预处理对小鼠肺缺血再灌注损伤的保护作用目的：探讨吸入不同时长的低浓度一氧化氮(NO)预处理对小鼠肺缺血再灌注损伤的影响和机制。背景：NO可以参与体内信号转导,炎症反应等多个环节,具有舒张血管和局部抗炎的功能。实验证明,吸入低浓度NO预处理对肺缺血再灌注损伤具有保护作用,但尚无关于NO预处理减轻肺缺血再灌注损伤最佳吸入时长的研究报道,本研究应用小鼠肺缺血再灌注损伤模型,探讨NO预处理吸入的最佳时长。方法和结果：6-8周雄性C57BL小鼠,分为空白组(S组),缺血再灌注损伤组(I/R组),NO预处理1min组(NO1-min组),NO预处理10min组(NO10-min组),NO预处理60min组(NO60-min组),分别于再灌注损伤后6h采集标本,检测肺动脉血氧分压(Pa02),肺组织干/湿重比(W/D),髓过氧化酶(MPO)活性,肺损伤组织学评价等指标；取NO预处理的最佳吸入时长,比较各组肺组织中Toll样受体2和4(TLR2/4)的基因及蛋白水平的表达,同时检测各组血清中TNF-a的表达变化。与I/R组比较,NO1-min组肺缺血再灌注损伤无改善(P>0.05)；NO10-min组与NO60-min组对肺缺血再灌注损伤保护作用相似(P>0.05);NO10-min组TLR2/4基因及蛋白水平表达较I/R组降低(P<0.05)；结论：短时长(10min)吸入低浓度NO预处理可以改善小鼠肺缺血再灌注损伤,但其保护程度不与NO预处理时长成正比；NO预处理可以抑制肺缺血再灌注损伤引起的TLR2/4表达的激活,减轻炎症反应。第二部分应用基因芯片技术检测Toll样受体信号通路在一氧化氮对小鼠肺缺血再灌注损伤保护过程中的变化目的：应用Real-Time PCR基因芯片技术检测Toll样蛋白受体信号通路在一氧化氮(NO)对小鼠肺缺血再灌注损伤保护作用过程中的变化,以明确NO肺保护的新作用机制。背景：吸入低浓度NO预处理对肺缺血再灌注损伤具有保护作用,在本文第一部分研究中发现吸入NO预处理的肺保护过程中,Toll样蛋白受体2/4的表达激活被明显抑制；功能性Real-Tune PCR基因芯片技术,其灵敏度,重复性,特异性均较第一代基因谱芯片技术有较大的提高和改进,因此我们应用该项技术,了解Toll样蛋白受体信号通路在NO预处理的肺保护过程中的变化。方法和结果：6-8周雄性C57BL小鼠,分为空白组(S组),缺血再灌注损伤组(I/R组),NO预处理10min组(NO10-min组),分别于再灌注损伤后6h采集标本(每组3只),应用Real-TimePCR基因芯片技术检测Toll样蛋白受体介导的信号转导相关的91个基因表达谱变化。结果显示,与I/R组相比,NO10-min组Toll样蛋白受体介导的信号转导相关的83个基因表达下调,其中Toll样蛋白受体1/2/4/8/9,衔接蛋白MyD88,效应分子IRAK1,下游通路及靶基因IKBKB、IRF3、NFKB2、NFRKB、 MAP3K1(MEKKK)、IFNB1、 NFKB1等31个基因表达下调具有统计学意义(P<0.05)。结论：在低浓度吸入NO预处理小鼠肺缺血再灌注损伤过程中,Toll样蛋白受体信号通路的激活被明显抑制,提示NO可以通过抑制Toll样蛋白受体信号通路的激活起到减轻肺损伤的作用。第三部分Toll样受体信号通路参与一氧化氮预处理对小鼠肺缺血再灌注损伤的保护作用目的：应用基因敲除小鼠(MyD88-/-),建立小鼠肺缺血再灌注模型,探讨Toll样蛋白受体信号通路是否参与一氧化氮(NO)对小鼠肺缺血再灌注损伤的保护作用及调节机制。背景：吸入低浓度NO预处理对肺缺血再灌注损伤具有保护作用,本实验组研究发现吸入NO预处理的肺缺血再灌注损伤保护过程中,Toll样蛋白受体信号通路的基因水平表达激活被明显抑制,其中作为该信号通路核心枢纽的MyD88分子,表达抑制明显。方法和结果：6-8周雄性C57BL小鼠MyD88+/+),分为空白组(S组),缺血再灌注损伤组(I/R组),NO预处理10mmin组(NO10-min组);6-8周雄性MyD88-/-基因敲除小鼠(以C57BL为遗传背景),分为空白组(S组),缺血再灌注损伤组(I/R组),NO预处理10min组(NO10-min组),分别于再灌注损伤后6h采集标本(每组5只),RT-PCR检测Toll样受体2/4,TRAF6,IRF3的基因水平表达变化,及ELISA检测TNF-a,IL-6的蛋白水平表达变化。结果显示,MyD88+/+及MyD88-/-组内,NO预处理均可以抑制Toll样受体2/4,TRAF6,IRF3基因的激活表达(P<0.05),并且炎症因子TNF-α,IL-6的蛋白表达显著减少(P<0.05)。结论：Toll样蛋白受体信号通路通过MyD88依赖及非依赖途径参与NO对小鼠肺缺血再灌注损伤的保护作用。更多还原

【Abstract】 PartⅠ Short Term of Nitric Oxide Inhalation Protect Against the Mouse Lung Ischemia-Reperfusion InjuryObjective:To investigate the effect of inhaled nitric oxide (NO) preconditioning with light concentration and different terms on lung ischemia-reperfusion injury in mice.Background:NO can participate in the signal transduction, inflammation and other links. It has the function of diastolic blood vessels and topical anti-inflammatory. Inhaled with low concentration NO preconditioning has protective effect on lung ischemia/reperfusion injury. This study used lung ischemia/reperfusion model in mice and investigated the best length of inhaled NO pretreatment.Methods and Results:Specific pathogen-free C57BL mice (male,6-8weeks old) were divided into five groups:Sham (S) group, Ischemia/Reperfusion (IR) group, NO1-min preconditioning group (NO1-min), NO10-min preconditioning group (NO10-min), NO60-min preconditioning group (NO60-min). The changes of PaO2, wet-dry weight ratio (W/D), myeloperoxidase (MPO) in the injury lung were measured after60minutes of left lung ischemia. The changes of TLR2/4activations and plasma TNF-α were measured in this procedure. Compared with IR group, PaO2increased and W/D, MPO decreased evidently in NO10-min group (P<0.05). The changes in NO60-min group were similar with NO10-min group (P>0.05). There was no difference between NO1-min and IR group (P>0.05). In NO10-min group, the expressions of TLR2/4mRNA and TLR2/4proteins were diminished and TNF-a concentrations were decreased.Conclusion:Short term (10min) of inhalation NO protected lung IR injury in mice. But the protective effect of NO was not increased with the longer term of inhalation NO. Inhaled NO could inhibit the activations of TLR2/4in the lung after IR injury. Part Ⅱ Application of Real-Time PCR Gene Chip Technology to Detect the Role of Toll-like Protein Receptor Signaling Pathways in the Protective Effect of Nitric Oxide on Lung Ischemia-Reperfusion Injury in MiceObjective:Application of Real-Time PCR gene chip technology to detect toll-like protein receptor signaling pathways in nitric oxide (NO) on pulmonary ischemia-reperfusion injury in mice protection in the process of change, in order to understand the mechanism of NO lung protection.Background:Low concentration NO preconditioning has protective effect on pulmonary ischemia-reperfusion injury. In the first part of our studies found that lung protection during the process of the pretreatment of inhaled NO, Toll2/4expression activation was significantly inhibited; Functional Real-Time PCR, gene chip technology to Superarray companies in USA, the second generation of the gene chip, function classification has a high sensitivity, linear range wide good repeatability high resolution, high specificity. We used the technology to understand the toll-like receptor protein signaling pathways in the process of pretreatment with NO of lung protection.Methods and Results:6-8weeks male C57BL mice, divided into Sham group (S group), Ischemia-Reperfusion group (I/R group),10min NO pretreatment group (NO10-min group). Using Real-Time PCR gene chip technology to detect toll-like receptor mediated signal transduction protein gene expression patterns change, according to the results of91compared with I/R group, the NO10-min group toll-like receptor mediated signal transduction protein gene expression by80, including toll-like receptor proteins1/2/4/8/9, cohesion MyD88protein, the effect of molecular IRAK1, downstream pathway and target genes IKBKB, IRF3, NFKB2(P<0.05).Conclusion:At low concentrations of inhaled NO pretreatment in the process of lung ischemia-reperfusion injury in mice, the activations of toll-like receptor signaling pathways were significantly inhibited, NO can inhibit toll-like receptor signaling pathways to reduce lung injury. Part Ⅲ Toll-like receptors signaling pathways involved in pretreatment of nitric oxide in mice lung ischemia-reperfusion injury of protectionObjective:Application of knockout mice (MyD88-/-), lung ischemia-reperfusion in mice model was established, exploring toll-like protein receptor signaling pathways are involved in nitric oxide (NO) on pulmonary ischemia-reperfusion injury in mice.Background:Low concentration NO preconditioning has protective effect on pulmonary ischemia-reperfusion injury, this experimental study found that inhaled NO pretreatment of lung ischemia-reperfusion injury protection process, toll-like protein receptor signaling pathways of gene expression level activation was significantly inhibited, which as the core hub of MyD88signaling pathway molecules, expression inhibited significantly.Methods and Results:6-8weeks (MyD88+/+) of male C57BL mice, divided into blank group (group S), ischemia reperfusion group (I/R group),10min NO pretreatment group (NO10-min);6-8weeks male MyD88-/-knockout mice (C57BL as genetic background), divided into blank group (group S), ischemia reperfusion group (I/R group),10min NO pretreatment group (NO10-min), respectively, at6h after reperfusion, RT-PCR detection by2/4toll-like receptors, TRAF6, IRF3level of gene expression changes, and ELISA to detect TNF alpha, IL-6level of protein expression changes. Results show that the MyD88+/+and MyD88-/-within the group, NO pretreatment can inhibit toll-like receptors by2/4, TRAF6, IRF3the activation of gene expression (P<0.05), and inflammatory factor TNF alpha, IL-6protein expression significantly were reduced (P<0.05).Conclusion:Toll-like receptor signaling pathways through the MyD88-dependent pathway and MyD88-undependent pathway to participate in the NO in mice lung ischemia-reperfusion injury of protection.更多还原