【作者】 游志坚；

【导师】 姚尚龙；

【作者基本信息】 华中科技大学 ， 麻醉学， 2007， 博士

【摘要】 第一部分兔单肺通气模型的建立及三种方法的比较实验一兔单肺通气模型的建立目的建立兔单肺通气实验模型。方法健康日本大耳白兔10只,麻醉后仰卧位固定。右侧股静脉建立静脉通道,右侧股动脉置管监测动脉血压并采集动脉血标本;行气管切开并插入自制双腔气管导管实施机械通气,呼吸机通气参数为:FiO2 1.0 , RR 40/min,VT 10 ml/kg。以听诊法仔细调整自制双腔气管导管位置,并采用气泡试验法检测左侧气管膨大处的密闭效果。将动物调整为左侧卧位后,钳闭左侧管腔建立右肺单肺通气模型,并以纤支镜直接观察左侧肺叶呼吸运动是否停止并萎陷。单肺通气2h后,松开左侧管腔恢复双肺通气。在单肺通气前、单肺通气后30min和恢复双肺通气30min三个时间点测血压、心率、气道压和动脉血气分析。结果实验中单肺通气效果满意,操作方便,血流动力学稳定,各时点动脉血气变化规律与临床一致。结论采用自制双腔气管导管可以建立日本大耳白兔单肺通气模型。此模型简单方便,成功率高,适用于研究单肺通气相关的肺损伤和低氧血症的病理生理机制及其防治策略。实验二三种兔单肺通气模型的比较目的采用三种方法建立兔单肺通气模型并比较其效果。方法日本大耳白兔30只,随机分为3组(即A、B、C组)各10个,分别采用自制双腔气管导管法、左支气管结扎法和插管过深法。呼吸机通气参数为:FiO2 1.0 , RR 40/min,VT 10 ml/kg。单肺通气2h后恢复双肺通气。记录各组单肺通气实施的一次成功率、总成功率、从气管切开开始到单肺通气实施所需要的时间、动物失血量。结果各组进入实验的动物数分别为10、6、8只。与B、C组比较,A组一次成功率和总成功率高,所需时间明显较少(P<0.01),且出血量明显少于B组(P<0.01)。结论采用自制双腔气管导管能迅速有效的建立单肺通气模型,优于左支气管结扎法和插管过深法,是用于研究与单肺通气相关病理生理机制的理想模型。第二部分单肺通气后两侧肺损伤特点及不同时间的影响目的建立兔单肺通气模型后,观察两侧肺损伤的特点以及不同时间的影响,为后续的研究摸索条件。方法健康日本大耳白兔24只,随机分为4组:对照组(C组,n=6)双肺通气2h;右侧单肺通气组(O1组、O2组及O3组,n=6)单肺通气时间分别为1h、2h、3h,随后恢复双肺通气1h。在实验结束前开胸,分别由两侧肺静脉同时抽取肺静脉血、由股动脉抽取动脉血做血气分析并测定氧合指数;采用颈动脉快速放血处死动物,将各组左右肺分开处理。检测各组肺组织病理改变,并计算肺损伤评分;检测各肺组织湿干重比值、MPO、MDA、SOD。结果⑴C组左右侧肺、O1组右侧肺组织结构形态完整,肺泡内无明显渗出液,肺间质无明显水肿和炎症;O1组左侧肺、O2组右侧肺和O3组右侧肺肺泡内可见渗出液,肺间质增厚并可见炎性细胞浸润;O2组左侧肺、O3组左侧肺肺泡内可见渗出液,部分肺泡腔萎陷不张或充血,肺间质增厚并可见大量炎性细胞浸润。肺损伤评分:C组左右侧相比较差异无统计学意义(P﹥0.05),另三组组内左右侧比较则左侧明显增高(P<0.01);各组间右侧比较表现为逐步升高,其中O3组右侧与O1组右侧相比较明显增高(P<0.05);各组间左侧相比较亦表现为逐步升高,其中O2组左侧、O3组左侧与O1组左侧相比较明显增高(P<0.01),O1组左侧与C组左侧相比较明显增高(P<0.01)。⑵各组氧合指数比较,C组左右侧相比较差异无统计学意义(P﹥0.05),另三组组内左右侧比较则左侧明显下降(P<0.01);各组间右侧比较表现为逐步降低,其中O3组右侧与O1组右侧相比较明显降低(P<0.01);各组间左侧相比较亦表现为逐步降低,其中O2组左侧、O3组左侧与O1组左侧相比较明显下降(P<0.01),O1组左侧与C组左侧相比较明显降低(P<0.01)。动脉血气所测得氧合指数表现为逐步降低,O2组、O3组与O1组相比较明显下降(P<0.01),O1组与C组相比较明显降低(P<0.01)。⑶各组湿/干重比、MPO活性和MDA的变化表现为C组组内左右侧相比较差异无统计学意义(P﹥0.05),O1组、O2组和O3组组内左右侧比较则左侧明显增高(P<0.01);各组间右侧比较表现为逐步升高,其中O3组右侧与O1组右侧相比较明显增高(P<0.01),O1组右侧与C组右侧相比较明显增高(P<0.01);各组间左侧相比较亦表现为逐步升高,其中O2组左侧、O3组左侧与O1组左侧相比较明显增高(P<0.01),O1组左侧与C组左侧相比较明显增高(P<0.01)。⑷各组SOD值的变化表现为C组和O1组组内左右侧相比较差异无统计学意义(P﹥0.05),O2组和O3组组内左右侧比较则左侧明显降低(P<0.01);各组间右侧比较表现为逐步降低,其中O3组右侧与O1组右侧相比较明显降低(P<0.01),O1组右侧与C组右侧相比较明显降低(P<0.01);各组间左侧相比较表现为逐步降低,其中O2组左侧、O3组左侧与O1组左侧相比较明显降低(P<0.01),O1组左侧与C组左侧相比较明显降低(P<0.01)。结论长时间单肺通气可以导致急性肺损伤,这种损伤是不均一性的,损伤的程度与单肺通气时间相关,且非通气侧肺要比通气侧肺损伤严重。第三部分单肺通气所致肺损伤中NF-κB的作用目的通过建立兔单肺通气模型,观察NF- B在单肺通气肺损伤中的作用及对炎性因子TNF-和ICAM-1的影响,以探讨单肺通气肺损伤的机制。方法健康日本大耳白兔18只,随机分为3组:TLV组、OLV组和PDTC组,各6只。TLV组实施双肺机械通气3h。OLV组采用自制双腔气管导管实施右侧单肺通气2h后恢复双肺通气1h。PDTC组在实施单肺通气前由静脉输入PDTC50mg/kg,其后操作与OLV组相同。在实验结束前由股动脉抽取动脉血做血气分析测定氧合指数。各组左右肺分开处理。光学显微镜下观测各组肺组织病理改变,并计算肺损伤评分;检测各肺组织湿/干重比值、MPO、MDA、SOD; Western Blot检测肺细胞核内NF-κB p65蛋白和胞浆内IκB-α的表达水平;EMSA检测肺组织细胞NF-κB的活性;ELISA测TNF-α和ICAM-1的含量,并用RT-PCR检测肺组织中TNF-α和细胞粘附分子(ICAM-1)基因mRNA的表达水平。结果⑴病理学改变TLV组左右侧肺组织结构形态完整;OLV组右侧肺泡内可见渗出液,肺间质增厚并可见炎性细胞浸润,而其左侧肺泡内可见渗出液,部分肺泡腔萎陷不张或充血,肺间质增厚并可见大量炎性细胞浸润;PDTC组右侧肺组织与OLV组右侧肺组织相似,而其左侧肺较OLV组明显改善,但肺泡内仍可见渗出液,肺间质增厚及炎性细胞浸润。肺损伤评分,OLV组和PDTC组左右侧比较则左侧明显增高(P<0.01);各组间比较,OLV组和PDTC组双侧均高于TLV组(P<0.01);PDTC组左侧明显低于OLV组(P<0.01)。⑵氧合指数与TLV组比较,OLV组和PDTC组均下降(P<0.01),且PDTC组与OLV组相比明显上升(P<0.05)。⑶各组湿/干重比、MPO活性和MDA的变化表现为OLV组和PDTC组组内左右侧比较则左侧明显增高(P<0.01);各组间比较,OLV组和PDTC组双侧均高于TLV组(P<0.01);与OLV组比较,PDTC组双侧均明显降低(P<0.01或P<0.05)。⑷各组SOD值的变化表现为OLV组左右侧比较则左侧明显降低(P<0.05);各组间比较,OLV组和PDTC组双侧肺均低于TLV组(P<0.01);PDTC组左侧明显高于OLV组(P<0.05)。⑸NF- B的含量与活性变化表现为OLV组和PDTC组左右侧比较左侧明显增高(P<0.01);各组间比较,OLV组和PDTC组双侧肺均高于TLV组(P<0.01),且与OLV组相比较,PDTC组明显降低(P<0.01或P<0.05)。I B-的含量变化表现为OLV组和PDTC组左右侧比较左侧明显增高(P<0.01);各组间比较,OLV组和PDTC组双侧肺均低于TLV组(P<0.01),且PDTC组高于OLV组(P<0.05)。⑹TNF-与ICAM-1的含量及mRNA的表达变化表现为OLV组和PDTC组左右侧比较左侧明显增高(P<0.01或P<0.05);各组间比较,OLV组和PDTC组双侧肺均高于TLV组(P<0.01或P<0.05),且与OLV组相比较,PDTC组明显降低(P<0.01)。结论单肺通气导致的肺损伤中NF- B途径被激活,促进炎症因子的合成,在单肺通气肺损伤中起到了重要作用,而采用特异性NF- B抑制剂PDTC可以抑制NF- B途径的激活,减少炎症因子的合成,减轻肺损伤程度。第四部分肺保护性通气和降低吸入氧浓度对单肺通气肺损伤的影响目的研究在兔OLV模型中采用肺保护性通气,同时降低吸入氧浓度,观察其对肺部炎症反应和NF-κB表达的影响,探讨OLV时合理的通气模式。方法健康日本大耳白兔18只,随机分为3组:A组、B组和C组,各6只。采用自制双腔气管导管建立单肺通气模型,右单肺通气2h后恢复双肺通气1h。呼吸机通气参数为:A组, RR 40/min,VT 10 ml/kg,FiO2100%;B组,RR 40/min,VT 7 ml/kg,PEEP 5cmH2O,FiO2 100%;C组,RR 40/min,VT 7 ml/kg,PEEP 5cmH2O,FiO2 60%。分别在OLV前、OLV后30min和恢复双肺通气(TLV)30min三个时间点做动脉血气分析并计算氧合指数。各组左右肺分开处理。光学显微镜下观测各组肺组织病理改变,并计算肺损伤评分;检测各肺组织湿/干重比值、MPO、MDA、SOD; Western Blot检测肺细胞核内NF-κB p65蛋白和胞浆内IκB-α的表达水平;EMSA检测肺组织细胞NF-κB的活性;ELISA测TNF-α和ICAM-1的含量, RT-PCR检测肺组织中TNF-α和ICAM-1mRNA的表达水平。结果⑴病理学改变A组右侧肺泡内可见渗出液,肺间质增厚并可见炎性细胞浸润,而其左侧肺泡内可见渗出液,部分肺泡腔萎陷不张或充血,肺间质增厚并可见大量炎性细胞浸润;B组右侧肺组织与A组右侧肺组织相似,而其左侧肺较A组明显改善;C组右侧与B组相似,而其左侧较B组轻。肺损伤评分,三组左右侧比较左侧明显增高(P<0.01);B组和C组双侧肺均低于A组(P<0.01),但B组与C组相比无统计学意义(P﹥0.05)。⑵氧合指数各组动物在各时点的pH值均在7.20以上,且PaCO2均小于50mmHg;三组动物氧合指数在各时点的变化表现为OLV30min后两组均显著下降(P<0.01),恢复TLV30min后上升,但仍低于OLV前(P<0.01)。三组动物在OLV前氧合指数无统计学意义(P﹥0.05);OLV30min后B组和C组高于A组(P<0.01或P<0.05);在恢复TLV30min后,B组和C组明显高于A组(P<0.01),且氧合指数B组和C组均大于300,而A组小于300。⑶各组湿/干重比、MPO活性和MDA的变化表现为组内左右侧比较左侧明显增高(P<0.01或P<0.05);各组间同侧比较,B组和C组均低于A组(P<0.01或P<0.05);C组左侧明显低于B组(P<0.05)。⑷各组SOD值的变化表现为左右侧比较左侧明显降低(P<0.05);各组间同侧比较,B组和C组均高于A组(P<0.01或P<0.05);C组左侧明显高于B组(P<0.05)。⑸NF- B的含量与活性变化表现为左右侧比较左侧明显增高(P<0.01);各组间同侧比较,B组和C组均低于A组(P<0.01),且与B组相比较,C组明显降低(P<0.05)。I B-的含量变化表现为左右侧比较左侧明显降低(P<0.01);各组间同侧比较,B组和C组均高于A组(P<0.01);C组左侧明显高于B组(P<0.05)。⑹TNF-与ICAM-1的含量及mRNA的表达变化表现为左右侧比较左侧明显增高(P<0.01或P<0.05);各组间同侧比较,B组和C组均低于A组(P<0.01),且与B组相比较,C组明显降低(P<0.05)。结论肺保护性通气+降低吸入氧浓度可以减少单肺通气时肺损伤程度,其作用机制可能与通过各种途径降低NF- B活化,减少炎症因子合成、炎症细胞聚集和氧化应激损伤有关。更多还原

【Abstract】 Part one Building one-lung ventilation model in rabbits and comparisons of three kinds of methodsTest one: Rabbit model of One-lung ventilation.Objective The purpose of this study was to build an experimental model of one lung ventilation (OLV) in rabbits.Method Ten rabbits weighing 2.3±0.2kg were anesthetized with ketamine (25 mg/kg, IM). A double-lumen endotracheal tube made by myself was inserted, and mechanical ventilation with 100% oxygen was used during the study. The ventilator settings were VT = 10 ml/kg, respiratory frequency = 40breaths/min and inspiratory-expiratory rate = 1:2. Mean arterial pressure, heart rate, pressure of air way and arterial blood gases were recorded in three phases: 5 minutes before OLV, 30 minutes after beginning OLV and 30 minutes after returning to two-lung ventilation (TLV). Result This rabbit model of OLV is convenient, satisfactory and safety. The changes of Mean arterial pressure, heart rate, pressure of air way and arterial blood gases were similar to clinical.Conclusion The rabbit model of OLV was easy to perform and could reduce the spending of animals and equipments. This model is suitable for the investigation of the pathophysiological mechanism and possible protective interventions to lung injury and hypoxia induced by OLV.Test two: A comparison of three kinds of One-lung ventilation models in rabbits. Objective The purpose of this study was to compare three kinds of one-lung ventilation (OLV) models in rabbits.Method 30 rabbits weighing 2.3±0.2kg were anesthetized with ketamine (25 mg/kg, IM) and were ventilated via tracheotomy tubes at a rate of 40/min with a tide volume of 10 ml/kg with pure oxygen and divided randomly into three groups. In group A (n=10) OLV was carried out by a artificial double-lumen endotracheal tube. In group B thoracotomy was performed and the left main bronchus was completely clamped with a clip, so OLV was carried out. In group C tracheotomy tube was pushed into the right main bronchus to perform OLV. The successful rates of first time and overall, the time from tracheotomy beginning to OLV beginning and volume of bleeding were recorded. The rabbit’s tracheal was measured.Result In group A, the success rates of first time and overall were higher than group B and group C. The numbers of three groups coming into experiment were respectively 10, 6, 8. In group A the time from tracheotomy beginning to OLV beginning were significantly than group B and group C(P<0.01). To compared with group B the bleeding volume was significantly lower in group A (P<0.01).Conclusion The rabbit model of OLV performed by the artificial double-lumen endotracheal tube is convenient, satisfactory and safe. This model is suitable for the investigation of the pathophysiological mechanism associated with OLV. Part 2 Character of one-lung ventilation induced lung injury and the effect of different durations on lung injury degrees of bilateral lungsObjective To investigate the character of one-lung ventilation induced lung injury and the effect of different durations on lung injury degrees of bilateral lungs.Methods 24 white Japanese rabbits were divided into 4 groups (n = 6). The control group performed two-lung ventilation for 2h. The groups of O1、O2 and O3 performed one-lung ventilation for 1h、2h and 3h respectively, which were followed with two-lung ventilation for 1h. Lung separation was achieved with a artificial double-lumen tube. The blood samples were drawn from femoral artery、the right pulmonary vein and the left one to get the oxygenation indexes. Light microscopic evaluations were performed to get the lung injury scores. Wet/dry ratios (W/D), MDA、SOD、myeloperoxidase (MPO) activity were measured with corresponding methods.Results Lung injury scores were significant differences in left lung compared to right lung in the groups of O1、O2 and O3, though no significant differences were noted in the group C. Comparison of the right lungs of the four groups documented significant increase of lung injury scores. Compared with O1 group, Lung injury scores in O3 group was significantly elevated(P<0.05). The changes in the left lung were similar and more severe to the right ones. Compared with C group, Lung injury scores in O1 group was significantly elevated(P<0.01). Compared with O1 group, Lung injury scores in O2 and O3 groups were significantly elevated(P<0.01). Similar changes of oxygenation index、W/D、MDA and MPO activity were found in all the groups, which were significant differences in left lung compared to right lung in the groups of O1、O2 and O3, though no significant differences were noted in the group C. Comparison of the right lungs of the four groups documented significant increase of lung injury scores. Compared with O1 group, O3 group was significantly elevated(P<0.05). The changes in the left lung were similar and more severe to the right ones. Compared with C group, O1 group was significantly elevated(P<0.01). Compared with O1 group, O2 and O3 groups were significantly elevated(P<0.01). SOD was significant differences in left lung compared to right lung in the groups of O2 and O3. Compared with C group, SOD in right and left lung of O1 group were significantly decreased(P<0.01).SOD in right of O3 group was less than O1 group(P<0.01). SOD in left of O2 and O3 group were less than O1 group(P<0.01).Conclusion Long time OLV could provoke acute lung injury, which was inhomogeneous. The degrees of lung injury were related to OLV duration and were more severe in the non-ventilated lung.Part 3 Effects of nuclear factor- B on one-lung ventilation induced lung injuryObjective To study the changes of nuclear factor- B activation and expression of cytokines in lung tissues after one lung ventilation on the rabbit models.Methods 18 white Japanese rabbits were divided into 3 groups (n = 6). The TLV group performed two-lung ventilation for 3h. The OLV group performed one-lung ventilation for 2h, which were followed with two-lung ventilation for 1h. Lung separation was achieved with a artificial double-lumen tube. The PDTC group were pretreated with pyrrolidine dithiocarbamate (PDTC) (50mg/kg, IV.) prior to one lung ventilation. The blood samples were drawn from femoral artery to get the oxygenation indexes. Light microscopic evaluations were performed to get the lung injury scores. Wet/dry ratios (W/D), MDA、SOD、myeloperoxidase (MPO) activity were measured with corresponding methods. NF-κB p65 and IκB-αwere detected with Western Blot. NF-κB activity was assayed with EMSA. TNF-αand ICAM-1 in lung tissues were detected with ELISA. The expression of TNF-αand ICAM-1 mRNA in lung tissue was measured by RT-PCR.Results (1)Lung injury scores were significant differences in left lung compared to right lung in the groups of OLV and PDTC(P<0.01). Compared with TLV group, Lung injury scores in OLV and PDTC groups were significantly elevated(P<0.05). Compared with OLV group, Lung injury scores in PDTC group was significantly decreased(P<0.01). (2) Compared with TLV group, the oxygenation of OLV and PDTC group were significantly decreased(P<0.01). Compared with OLV group, the oxygenation of PDTC group were significantly increased(P<0.05). (3) W/D、MDA and MPO activity were significant differences in left lung compared to right lung in the groups of OLV and PDTC(P<0.01). Compared with TLV group, OLV and PDTC groups were significantly elevated(P<0.01). Compared with OLV group, PDTC group was significantly decrease(dP<0.01 or P<0.05). (4)SOD was significant differences in left lung compared to right lung in OLV group(P<0.01). Compared with TLV group, OLV and PDTC groups were significantly decreased(P<0.01). Compared with OLV group, the left lung of PDTC group was significantly increased(P<0.01 or P<0.05). (5) NF-κB activity and NF-κB p65 were significant differences in left lung compared to right lung in the groups of OLV and PDTC(P<0.01). Compared with TLV group, OLV and PDTC groups were significantly elevated(P<0.01). Compared with OLV group, PDTC group was significantly decreased(P<0.01 or P<0.05). I B- was significant differences in left lung compared to right lung in the groups of OLV and PDTC(P<0.01). Compared with TLV group, OLV and PDTC groups were significantly decreased(P<0.01). Compared with OLV group, PDTC group was significantly increased(P<0.01 or P<0.05). (6) The quantity of TNF- and ICAM-1 and the expression of TNF- and ICAM-1mRNA were significant differences in left lung compared to right lung in the groups of OLV and PDTC(P<0.01 or P<0.05). Compared with TLV group, OLV and PDTC groups were significantly elevated(P<0.01 or P<0.05). Compared with OLV group, PDTC group was significantly decrease(dP<0.01 or P<0.05). Conclusion NF- B activation probably play an important role in lung injury caused by one-lung ventilation.Part 4 Effects of lung protective ventilation strategy and the fraction of inspired oxygen on lung injury degree in one-lung ventilation modelsObjective To study the effects of lung protective ventilation strategy and the fraction of inspired oxygen on lung injury degree in one-lung ventilation models.Methods 18 white Japanese rabbits were performed one-lung ventilation for 2h, which were followed with two-lung ventilation for 1h. Lung separation was achieved with a artificial double-lumen tube. The rabbits were divided into 3 groups (n = 6): (1) normal control group (A group): RR 40/min, VT 10 ml/kg and FiO2 100%; (2) protective ventilation group (B group): RR 40/min, VT 7 ml/kg, PEEP 5cmH2O and FiO2 100%; (3) protective ventilation and lower FiO2 group (C group): RR 40/min, VT 7 ml/kg, PEEP 5cmH2O and FiO2 60%. The blood samples were drawn from femoral artery to get the oxygenation indexes in three phases: 5 minutes before OLV, 30 minutes after beginning OLV and 30 minutes after returning to TLV. Light microscopic evaluations were performed to get the lung injury scores. Wet/dry ratios (W/D), MDA、SOD、myeloperoxidase (MPO) activity were measured with corresponding methods. NF-κB p65 and IκB-αwere detected with Western Blot. NF-κB activity was assayed with EMSA. TNF-a and ICAM-1 in lung tissues were detected with ELISA. The expression of TNF-a and ICAM-1 mRNA in lung tissue was measured by RT-PCR.Results (1)Lung injury scores were significant differences in left lung compared to right lung in all groups(P<0.01). Compared with A group, B and C groups were significantly decreased(P<0.05). (2) After returning to TLV, Compared with A group, the oxygenation indexes of B and C groups, which were greater than 300 while that of group A was less than 300, were significantly increased(P<0.01). (3) W/D、MDA and MPO activity were significant differences in left lung compared to right lung in all groups(P<0.01 or P<0.05). Compared with A group, B and C groups were significantly decreased(P<0.01). Compared with B group, the left lung of C group was significantly decreased(P<0.05). (4)SOD was significant differences in left lung compared to right lung in all group(sP<0.01). Compared with A group, B and C groups were significantly increased(P<0.01 or P<0.05). Compared with B group, the left lung of C group was significantly increased(P<0.01 or P<0.05). (5) NF-κB activity and NF-κB p65 were significant differences in left lung compared to right lung in all groups(P<0.01). Compared with A group, B and C groups were significantly deceased(P<0.01). Compared with B group, C group was significantly decreased(P<0.05). I B- was significant differences in left lung compared to right lung in all group(sP<0.01). Compared with A group, B and C groups were significantly increased(P<0.01). Compared with B group, the left lung of C group was significantly increased(P<0.05). (6) The quantity of TNF- and ICAM-1 and the expression of TNF- and ICAM-1mRNA were significant differences in left lung compared to right lung in all groups(P<0.01 or P<0.05). Compared with A group, B and C groups were significantly decreased(P<0.01 or P<0.05). Compared with B group C group was significantly decreased(P<0.05).Conclusion OLV with lung protective ventilation strategy and 60% inspired oxygen can attenuate lung injury by decreasing the activity of NF- B.更多还原