部分液体通气对肺表面活性物质的影响及其抗炎机制的探讨
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摘要
前言
急性肺损伤(ALI)是严重感染、创伤、休克等发生后常见的并发症，以肺间质水肿和实质细胞受损为主要病理表现，往往伴随着顽固性低氧血症和难以控制的炎症反应，所以提高受损肺泡的通气和换气能力，抑制炎症的进行性发展是治疗ALI必须面对的问题。用具有高氧和高二氧化碳溶解度的氟碳化合物(PFC)进行部分液体通气(PLV)，可明显地提高ALI后机体的血氧分压，增加肺的顺应性，并被认为可以抑制炎性细胞因子的分泌。
肺表面活性物质(PS)分泌不足是肺损伤低氧血症的主要原因。PS分泌不足造成肺泡气-液交界面表面张力增高，肺泡进行性萎陷，形成肺不张、通气／血流比失调，影响肺泡的通气和换气能力。所以保护残存肺泡的生理功能，增加PS的分泌，将有助于改善机体的低氧血症。目前研究认为，PLV降低肺泡表面张力，增加肺脏的顺应性，防止呼气末肺泡萎陷，提高机体血氧分压的机理是：①低张力系数的PFC本身就相当于表面活性物质，且与PS不同，不能被肺损伤时大量渗出的血浆蛋白所灭活；②呈液态的PFC使肺泡气-液交界面变成了液-液面，表面张力大大下降，同时起到了“液体PEEP”的作用，防止受损肺泡在呼气末萎陷；③密度较大的PFC在重力作用下优先进入下垂肺野增加肺泡内压力和肺泡周围血管阻力，使更多的血液流向肺上部，增加供氧充分区域的血流，优化了通气／血流比。但PLV是否会有助于肺泡Ⅱ型上皮细胞分泌PS，尤其是对PS的活性成分及相关蛋白的影响尚不清楚。
各种感染和非感染因素所引起的ALI往往伴随着难以控制的炎症反应，而这种炎性细胞之间的信号传导紊乱、失控是ALI发展为ARDS的基础。由于特殊的解剖结构和生理特性，肺脏往往成为全身炎性反应综合征(SIRS)这一病理过程所累及的首位靶器官。严重感染和创伤时，大量的中性粒细胞、单核细胞在肺内趋化、聚集、激活并释放炎性细胞因子，使白细胞和血管内皮细胞发生粘附，破坏肺泡和毛细血管膜，使之通透性增加，形成ALI的病理基础。用PFC进行PLV治疗ALI，与常规呼吸机支持治疗相比，
可减轻肺组织的炎症破坏，抑制中性粒细胞和单核一巨噬细胞在肺内的聚
集，减少TNF一a、IL一lp等炎性细胞因子的释放，这表明PLV具有一定的抗
炎作用，但PLV的抗炎机制尚不清楚。
本课题在总结国内外最新研究成果的前提下，针对ALI的发病机制，探
讨PLV治疗AU的理论基础。本研究分为三个部分:一、部分液体通气对
油酸诱导的兔急性肺损伤肺表面活性物质的影响;二、部分液体通气对兔肺
损伤后表面活性物质相关蛋白A的影响;三、氟碳化合物对脂多糖刺激中
性粒细胞NF一KB活化的影响。
实验材料与方法
一、实验方法
(一)第一部分
1.动物预处理取24只日本大白兔腹腔内注射氯胺酮10Om岁kg麻醉，
开放耳缘静脉，气管切开，给予洋库滨按0.4m扩kg后，插人气管导管连接
西门子gooC呼吸机，进行容量控制通气。吸入氧浓度为1 .0，频率35次/
min，吸气时间:呼气时间二1:2，设置呼气末正压(PEEP)为5 cm HZO，调节
潮气量使PaCOZ在35礴smm Hg之间。右股动脉插管，连接日本光电多导生
理监测仪，连续描记动脉压、心率变化。行中心静脉穿刺，在波形的监测下
送导管入右心室。待上述操作结束后20分钟，测量血气、平均动脉压
(MAP)、心率(HR)和吸气相峰压(PIP)的数据为基础值。
2.急性肺损伤模型的建立测定基础值后，通过中心静脉在30分钟内
缓慢滴注油酸150林岁kg，当paOZ/FIO:<300，即认为ALI造模成功。
3.实验动物分组将24只成年日本大白兔随机分为3组，每组8只。
C组:正常对照组，不造成肺损伤，单纯行机械通气，呼吸参数设置同
上。
CMV组:油酸肺损伤机械通气组，呼吸参数设置同上。
PLV组:油酸肺损伤部分液体通气组，呼吸参数设置同上。在保持机械
通气的基础上，10分钟内向气管导管内灌注3俨C的PFC 18mFkg。因PFC
具有挥发性，故每小时追加PFcs而。
实验过程中，每小时静脉输注浮库澳钱0.Zm扩kg、氯胺酮20m扩kg和
乳酸钠林格液6IllFkg，并于油酸后的第1小时及治疗后的1、2、4小时测量
动脉血气。
4.观察指标及方法
(l)描记压力一容积曲线实验结束后，在麻醉状态下处死动物，立即开
胸，完整暴露心肺器官，参照陈卫民报道的方法，将三通管分别与气管导管、
自制U形压力计和20d的注射器相连接，缓慢向肺内注人空气，使气道内
压力以5 cm HZO为一个等级逐渐加到40 cm H20，各压力点稳定20秒后记
录充气体积。选用SPSS10.O软件描记压力一容积曲线。
(2)右支气管灌洗液(BALF)中细胞计数和双饱和磷脂酞胆碱(DP-
PC)、总蛋白(TP)、大、小聚集体(LA、SA)分析
①细胞计数
各取BA廿4nil，由同一专业人员显微镜镜检、涂片，计数红细胞
(RBC)、中性粒细胞(PMN)和巨噬细胞(AM)绝对值。
②DPPC的测量
各取去除细胞、碎片、PFC后的BALF 4nil，加人三氯甲烷一甲醇溶液(2:
1)12nil。在快速旋转震荡器上混均1分钟后，80飞离心巧分钟。将下层
含磷脂的三氯甲烷溶液放人10血玻璃试管内，37“c水浴，氮气吹干
Acute lung injury is the common complication after severe infection, trauma and shock, etc. Its main pathological manifestation is pulmonary mesenchyma e-dema and parenchyma injury, and it is often accompanied with progressive refractory hypoxia and inflammation. Therefore, to protect the injured alveolar and to inhibit the progressive development of inflammation become the essential problem when treating All. Partial liquid ventilation using perfluorocarbon (PFC) with high oxygen and high carbon dioxide solubility can obviously raise partial pressure of blood oxygen after ALI, increase lung compliance and inhibit secretion of inflammatory factor.
The main reason for hypoxia after lung injury is inadequate secretion of pulmonary surfactant ( PS) , which can cause increased surface tension of alveolar gas-liquid interface, progressive alveolar collapse, atelectasis and imbalance of ventilation perfusion ratio. So protection of physiological function of residual alveoli and increase PS secretion will improve hypoxia. The present studies show the mechanism of PLV increase pulmonary compliance and partial pressure of blood oxygen lies in the followings: 1. PFC of low tension itself is resemble surfactant and cannot be inactivated by the large amount of plasma protein after lung injury; 2. Because PFC is dense, it will gravitate to the dependent parts of the lungs primarily. The bulk of the liquid will re - open collapsed regions of lung, acting as liquid PEEP. 3. In PLV, some regions of lung, particularly the non - dependent regions, may be predominantly ventilated with gas. If pulmonary vessels in dependent lung regions are compressed by PFC in the alveo
li of these regions, blood flow could be diverted towards non-dependent, aerated lung. PLV thus improves matching of ventilation to perfusion. However, there
are no reports on whether PLV can help pneumocyte type II to secrete PS or whether PLV affects the component of PS.
Cascade-like inflammation caused by all kinds of infectious and noninfec-tious factors is the basis of ALI developed to ARDS. Treatment of ALI with PLV using PFC, compared with routine gas mechanical ventilation supported therapy, can inhibit intrapulmonary aggregation of neutrophil granulocyte and macropha-ges and decrease the concentration of inflammatory factor such as TNF-cuIL-lp in bronchoalveolar lavage fluid. Pathological research also proved PLV could relieve inflammatory damage of lung, but its exact mechanism is not clear yet. This project is based on the summary of latest researches, aiming at the etiology of ALI, to study the theory of treatment of ALJ using PLV. The study holds three parts: 1. The effect of partial liquid ventilation on the pulmonary surfactant in rabbit acute lung injury induced by oleic acid; 2. The effect of partial liquid ventilation on the pulmonary surfactant associated protein A in rabbit acute lung injury; 3. The effect of PFC on the activation of neutrophils NF-kB stimulated by lipopolysaccharide.
Materials and Methods
Parti
1. Animal Preparation
24 healthy New Zealand white rabbits, weighing between 2. 1 and 2. 6 kg, were were anesthetized with a 100 mg/kg of ketamine. After conventional intubation, a cuffed endotracheal tube was placed in the trachea and secured. Gas mechanical ventilation was initiated and a bolus injection of 0.4mg/kg of pancu-ronium was administered for paralysis. Mechanical ventilation was provided u-singa Siemens 900C ventilator with the following ventilatory settings: volume-controlled ventilation, tidal volume (TV) , 12 ml/kg; respiratory rate (RR) , 40 breaths/min; inspiratory to expiratory (I: E ) ratio, 1:2; the fraction of inspired oxygen (FIO2) , 1.0; and positive end-expiratory pressure ( PEEP) , 5 cm H2O. The right femoral artery was cannulated for arterial blood pressure monitoring and blood sampling. Subsequent anesthesia was maintained with an i. v. in-
jectionof ketamine 20 mg kg-1 h-1. Muscle relaxation was maintained by i. v. administration of pancuroniumbromide with continuous infusionO.2
急性肺损伤(ALI)是严重感染、创伤、休克等发生后常见的并发症，以肺间质水肿和实质细胞受损为主要病理表现，往往伴随着顽固性低氧血症和难以控制的炎症反应，所以提高受损肺泡的通气和换气能力，抑制炎症的进行性发展是治疗ALI必须面对的问题。用具有高氧和高二氧化碳溶解度的氟碳化合物(PFC)进行部分液体通气(PLV)，可明显地提高ALI后机体的血氧分压，增加肺的顺应性，并被认为可以抑制炎性细胞因子的分泌。
肺表面活性物质(PS)分泌不足是肺损伤低氧血症的主要原因。PS分泌不足造成肺泡气-液交界面表面张力增高，肺泡进行性萎陷，形成肺不张、通气／血流比失调，影响肺泡的通气和换气能力。所以保护残存肺泡的生理功能，增加PS的分泌，将有助于改善机体的低氧血症。目前研究认为，PLV降低肺泡表面张力，增加肺脏的顺应性，防止呼气末肺泡萎陷，提高机体血氧分压的机理是：①低张力系数的PFC本身就相当于表面活性物质，且与PS不同，不能被肺损伤时大量渗出的血浆蛋白所灭活；②呈液态的PFC使肺泡气-液交界面变成了液-液面，表面张力大大下降，同时起到了“液体PEEP”的作用，防止受损肺泡在呼气末萎陷；③密度较大的PFC在重力作用下优先进入下垂肺野增加肺泡内压力和肺泡周围血管阻力，使更多的血液流向肺上部，增加供氧充分区域的血流，优化了通气／血流比。但PLV是否会有助于肺泡Ⅱ型上皮细胞分泌PS，尤其是对PS的活性成分及相关蛋白的影响尚不清楚。
各种感染和非感染因素所引起的ALI往往伴随着难以控制的炎症反应，而这种炎性细胞之间的信号传导紊乱、失控是ALI发展为ARDS的基础。由于特殊的解剖结构和生理特性，肺脏往往成为全身炎性反应综合征(SIRS)这一病理过程所累及的首位靶器官。严重感染和创伤时，大量的中性粒细胞、单核细胞在肺内趋化、聚集、激活并释放炎性细胞因子，使白细胞和血管内皮细胞发生粘附，破坏肺泡和毛细血管膜，使之通透性增加，形成ALI的病理基础。用PFC进行PLV治疗ALI，与常规呼吸机支持治疗相比，
可减轻肺组织的炎症破坏，抑制中性粒细胞和单核一巨噬细胞在肺内的聚
集，减少TNF一a、IL一lp等炎性细胞因子的释放，这表明PLV具有一定的抗
炎作用，但PLV的抗炎机制尚不清楚。
本课题在总结国内外最新研究成果的前提下，针对ALI的发病机制，探
讨PLV治疗AU的理论基础。本研究分为三个部分:一、部分液体通气对
油酸诱导的兔急性肺损伤肺表面活性物质的影响;二、部分液体通气对兔肺
损伤后表面活性物质相关蛋白A的影响;三、氟碳化合物对脂多糖刺激中
性粒细胞NF一KB活化的影响。
实验材料与方法
一、实验方法
(一)第一部分
1.动物预处理取24只日本大白兔腹腔内注射氯胺酮10Om岁kg麻醉，
开放耳缘静脉，气管切开，给予洋库滨按0.4m扩kg后，插人气管导管连接
西门子gooC呼吸机，进行容量控制通气。吸入氧浓度为1 .0，频率35次/
min，吸气时间:呼气时间二1:2，设置呼气末正压(PEEP)为5 cm HZO，调节
潮气量使PaCOZ在35礴smm Hg之间。右股动脉插管，连接日本光电多导生
理监测仪，连续描记动脉压、心率变化。行中心静脉穿刺，在波形的监测下
送导管入右心室。待上述操作结束后20分钟，测量血气、平均动脉压
(MAP)、心率(HR)和吸气相峰压(PIP)的数据为基础值。
2.急性肺损伤模型的建立测定基础值后，通过中心静脉在30分钟内
缓慢滴注油酸150林岁kg，当paOZ/FIO:<300，即认为ALI造模成功。
3.实验动物分组将24只成年日本大白兔随机分为3组，每组8只。
C组:正常对照组，不造成肺损伤，单纯行机械通气，呼吸参数设置同
上。
CMV组:油酸肺损伤机械通气组，呼吸参数设置同上。
PLV组:油酸肺损伤部分液体通气组，呼吸参数设置同上。在保持机械
通气的基础上，10分钟内向气管导管内灌注3俨C的PFC 18mFkg。因PFC
具有挥发性，故每小时追加PFcs而。
实验过程中，每小时静脉输注浮库澳钱0.Zm扩kg、氯胺酮20m扩kg和
乳酸钠林格液6IllFkg，并于油酸后的第1小时及治疗后的1、2、4小时测量
动脉血气。
4.观察指标及方法
(l)描记压力一容积曲线实验结束后，在麻醉状态下处死动物，立即开
胸，完整暴露心肺器官，参照陈卫民报道的方法，将三通管分别与气管导管、
自制U形压力计和20d的注射器相连接，缓慢向肺内注人空气，使气道内
压力以5 cm HZO为一个等级逐渐加到40 cm H20，各压力点稳定20秒后记
录充气体积。选用SPSS10.O软件描记压力一容积曲线。
(2)右支气管灌洗液(BALF)中细胞计数和双饱和磷脂酞胆碱(DP-
PC)、总蛋白(TP)、大、小聚集体(LA、SA)分析
①细胞计数
各取BA廿4nil，由同一专业人员显微镜镜检、涂片，计数红细胞
(RBC)、中性粒细胞(PMN)和巨噬细胞(AM)绝对值。
②DPPC的测量
各取去除细胞、碎片、PFC后的BALF 4nil，加人三氯甲烷一甲醇溶液(2:
1)12nil。在快速旋转震荡器上混均1分钟后，80飞离心巧分钟。将下层
含磷脂的三氯甲烷溶液放人10血玻璃试管内，37“c水浴，氮气吹干
Acute lung injury is the common complication after severe infection, trauma and shock, etc. Its main pathological manifestation is pulmonary mesenchyma e-dema and parenchyma injury, and it is often accompanied with progressive refractory hypoxia and inflammation. Therefore, to protect the injured alveolar and to inhibit the progressive development of inflammation become the essential problem when treating All. Partial liquid ventilation using perfluorocarbon (PFC) with high oxygen and high carbon dioxide solubility can obviously raise partial pressure of blood oxygen after ALI, increase lung compliance and inhibit secretion of inflammatory factor.
The main reason for hypoxia after lung injury is inadequate secretion of pulmonary surfactant ( PS) , which can cause increased surface tension of alveolar gas-liquid interface, progressive alveolar collapse, atelectasis and imbalance of ventilation perfusion ratio. So protection of physiological function of residual alveoli and increase PS secretion will improve hypoxia. The present studies show the mechanism of PLV increase pulmonary compliance and partial pressure of blood oxygen lies in the followings: 1. PFC of low tension itself is resemble surfactant and cannot be inactivated by the large amount of plasma protein after lung injury; 2. Because PFC is dense, it will gravitate to the dependent parts of the lungs primarily. The bulk of the liquid will re - open collapsed regions of lung, acting as liquid PEEP. 3. In PLV, some regions of lung, particularly the non - dependent regions, may be predominantly ventilated with gas. If pulmonary vessels in dependent lung regions are compressed by PFC in the alveo
li of these regions, blood flow could be diverted towards non-dependent, aerated lung. PLV thus improves matching of ventilation to perfusion. However, there
are no reports on whether PLV can help pneumocyte type II to secrete PS or whether PLV affects the component of PS.
Cascade-like inflammation caused by all kinds of infectious and noninfec-tious factors is the basis of ALI developed to ARDS. Treatment of ALI with PLV using PFC, compared with routine gas mechanical ventilation supported therapy, can inhibit intrapulmonary aggregation of neutrophil granulocyte and macropha-ges and decrease the concentration of inflammatory factor such as TNF-cuIL-lp in bronchoalveolar lavage fluid. Pathological research also proved PLV could relieve inflammatory damage of lung, but its exact mechanism is not clear yet. This project is based on the summary of latest researches, aiming at the etiology of ALI, to study the theory of treatment of ALJ using PLV. The study holds three parts: 1. The effect of partial liquid ventilation on the pulmonary surfactant in rabbit acute lung injury induced by oleic acid; 2. The effect of partial liquid ventilation on the pulmonary surfactant associated protein A in rabbit acute lung injury; 3. The effect of PFC on the activation of neutrophils NF-kB stimulated by lipopolysaccharide.
Materials and Methods
Parti
1. Animal Preparation
24 healthy New Zealand white rabbits, weighing between 2. 1 and 2. 6 kg, were were anesthetized with a 100 mg/kg of ketamine. After conventional intubation, a cuffed endotracheal tube was placed in the trachea and secured. Gas mechanical ventilation was initiated and a bolus injection of 0.4mg/kg of pancu-ronium was administered for paralysis. Mechanical ventilation was provided u-singa Siemens 900C ventilator with the following ventilatory settings: volume-controlled ventilation, tidal volume (TV) , 12 ml/kg; respiratory rate (RR) , 40 breaths/min; inspiratory to expiratory (I: E ) ratio, 1:2; the fraction of inspired oxygen (FIO2) , 1.0; and positive end-expiratory pressure ( PEEP) , 5 cm H2O. The right femoral artery was cannulated for arterial blood pressure monitoring and blood sampling. Subsequent anesthesia was maintained with an i. v. in-
jectionof ketamine 20 mg kg-1 h-1. Muscle relaxation was maintained by i. v. administration of pancuroniumbromide with continuous infusionO.2
引文
1. Hirschl RB, Tooley R, Parent AC, et al. Improvement of gas exchange, pulmonary function, and lung injury with partial liquid ventilation. A study model in a setting of severe respiratory failure. Chest, 1995 , 108:500-508.
2. Fujino Y, Goddon S, Chiche JD, et al. Partial Liquid Ventilation Ventilates Better than Gas Ventilation. Am J Respir Crit Care Med, 2000,162: 650-657.
3．陈卫民，盛卓人，张秉钧，等．补充肺表面活性剂后胃液误吸大鼠的肺弹性及组织学变化．中国医科大学学报，1998，27：22-25．
4. Hohlfeld JM, Ahlf K, Enhorning G,, et al. Dysfunction of Pulmonary Surfactant in Asthmatics after Segmental Allergen Challenge. Am J Respir Crit Care Med, 1999,159 : 1803-1809.
5. Pelosi P, Crotti S, Brazzi L, et al. Computed tomography in adult respiratory distress syndrome: what has it taught us? Eur Respir J, 1996,9:1055-1062.
6. Quintel M, Hirschl RB, Roth H,et al. Computer tomographic assessment of perfluorocarbon and gas distribution during partial liquid ventilation for acute respiratory failure. Am J Respir Crit Care Med, 1998,158:249-255.
7. Leach, Greenspan, Rubenstein, et al. Partial liquid ventilation with perflubron in premature infants with severe respiratory distress syndrome: the LiquiVent Study Group. N Engl J Meal,1996,335:761-767.
8. Rotta, Steinhorn. Partial liquid ventilation reduces pulmonary neutrophil accumulation in an experimental model of systemic endotoxemia and acute lung injury. Crit Care Med, 1998, 26:1707-1715.
9．穆魁敏，刘秉锟，燕生，等．肺表面活性物质的检测．北京医科大学学报，1990，22：397-398．
10. Furue S, Kuwabara K, Mikawa K, et al. Crucial role of group IIA phospholipase A (2) in oleic acid - induced acute lung injury in rabbits. Am J Respir Crit Care Med, 1999,160 : 1292-1302.
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9．穆魁敏，刘秉锟，燕生，等．肺表面活性物质的检测．北京医科大学学报，1990，22：397-398．
10. Furue S, Kuwabara K, Mikawa K, et al. Crucial role of group IIA phospholipase A (2) in oleic acid - induced acute lung injury in rabbits. Am J Respir Crit Care Med, 1999,160 : 1292-1302.
11. Ueda, Ikegami, Jobe. Surfactant subtypes : in vitro conversion, in vivo function, and the effects of serum proteins. Am J Respir Crit Care Med, 1994,149 : 1254-1259.
12. Edwards YS. Stretch stimulation: its effects on alveolar type Ⅱ cell function in the lung. Comp Bioehem Physiol A Mol Integr Physiol,2001 ,129:245-260.
13. Obraztsov, Neslund, Kombrust,et al. In vitro cellular effects of perfluorochemicals correlate with their lipid solubility. Am J Physiol Lung Cell Mol Physiol,2000, 278 : L1018-L1024.
14. Chander A, Fisher AB. Regulation of lung sttrfaetant secretion. Am J Physiol, 1990 ,258 (6 Pt 1 ) : L241-1253.