【作者】 宋俊峰；

【导师】 孙波；

【作者基本信息】 复旦大学 ， 儿科学， 2008， 博士

【摘要】 背景:急性肺损伤（acute lung iniury,ALI）和急性呼吸窘迫综合征（acute respiratorydistress syndrome,ARDS）是由心源性以外的各种肺内外致病因素引起肺微血管和肺泡上皮损伤为主的肺部炎症综合征,临床表现为急性、进行性低氧性呼吸衰竭,是危及各个年龄段的临床危重症,在儿科重症监护病房（pediatric intensivecare unit,PICU）中病死率很高。难治性低氧血症是严重ALI/ARDS的突出临床特点。一旦出现难治性低氧血症,患儿都会接受肺保护性通气策略指导下的机械通气为主的呼吸支持治疗,辅之以特殊治疗技术如俯卧位、肺复张策略、吸入一氧化氮（inhaled nitric oxide,iNO）、外源性肺表面活性物质（surfactant,Surf）等。当患儿肺存在严重气体交换障碍、上述方法不能纠正低氧血症时,体外膜氧合（extracorporeal membrane oxygenation,ECMO）生命支持可作为最终治疗手段。应用人工肺原理,ECMO可在体外对静脉血进行氧合、排除CO₂,回输体内从而提供呼吸和循环功能的支持作用。同时,ECMO治疗的患儿可采用低浓度氧、低通气量、低通气频率的机械通气策略,最低限度地干预肺,使损伤肺得到休息,从而使损伤肺逐渐完成功能上的改善和病理上的修复。但ECMO治疗也有不良反应,在提供肺功能支持的同时也启动了机体的炎症反应。临床、实验研究显示,ECMO治疗常伴随出现外周血中性粒细胞减少,中性粒细胞活化,促炎介质（tumor necrosis factor（TNF）-α、interleukin（IL）-6、IL-8、IL-1β）释放,补体活化,肺部X线损伤评分增高等变化。ECMO启动的炎症反应可能导致肺脏的二次损伤,抑制ECMO所致炎症反应、减轻治疗后肺脏损伤将改善呼吸衰竭患儿的预后,提高ECMO救治技术生存质量。ALI/ARDS临床、实验研究显示iNO和/或Surf具有改善氧合、抑制肺内炎症反应的作用。iNO可通过减少肺内分流、优化通气、血流比例改善氧合,并具有抑制核转录因子（nuclear transcription factor,NF）-κΒ活性、减少促炎介质释放、降低粘附分子表达、抑制中性粒细胞粘附和迁移等抗炎作用。外源性Surf的应用可恢复、增强损伤肺的表面活性物质功能,同时具有抑制自由基生成、降低促炎细胞因子合成、抑制中性粒细胞活化等抗炎功能。另外,实验研究显示,联合应用iNO、Surf具有较单一应用iNO或Surf更好的抗炎疗效。但iNO、Surf联用能否减轻ECMO所诱导的炎症反应、减轻治疗后肺脏损伤、促进肺脏恢复等尚不清楚。因此,本研究对行ECMO治疗的ALI模型联用iNO、Surf以探讨二者的肺保护作用。目的:1.观察ECMO对幼猪0-24 h呼吸系统、血流动力学的影响,监测ECMO所致炎症反应;探讨幼猪长时间生存（168 h）后肺病理、生化变化;2.对行ECMO治疗的正常、ALI幼猪联用iNO、Surf在抑制ECMO所致炎症反应、减轻治疗后肺损伤及对长时间生存幼猪肺内生长因子、纤维化等方面的影响。方法:健康雄性幼猪（4-5周龄、体重9-14 kg）23只,经镇静麻醉后均给予气道插管和低潮气量（7-9 ml/kg）机械通气。ALI模型经静脉注射18-20μg/Kg脂多糖（1ipopolysaccharide,LPS）后机械通气4-8 h诱发形成。ALI判断标准:（1）动脉血氧分压/吸入氧浓度（PaO₂/FiO₂）≤300 mmHg;（2）呼吸系统动态顺应性（dynamic compliance,Cdyn）较基础状态下降30%以上。（3）并且在出现上述指标（0 h）及12、24 h肺病理切片显示大量炎症细胞浸润,肺间隔和肺泡腔水肿,肺泡萎馅,符合ALI的病理改变（n=3）。另20只动物随机分为5组（n=5）:VENT组:机械通气;VENOS组:机械通气,吸入NO（iNO）10 ppm,气道滴入猪肺Surf制剂50 mg/kg;ECMO组:机械通气,ECMO治疗;ENOS组:机械通气,ECMO治疗,联合应用iNO、Surf（参照VENOS组）。另有5只健康幼猪为NENOS组:健康幼猪行机械通气,ECMO治疗,联合应用iNO、Surf（参照VENOS组）。常规性补液、纠正低血压、酸中毒、电解质紊乱等。在非ECMO干预动物,调节FiO₂、呼吸频率保持PaO₂大于60 mmHg、PaCO₂介于35与45mmHg间。ECMO干预动物持续静脉滴注肝素保持活化凝血时间（activatedclotting time,ACT）介于180-220 s间,保持ECMO流量为70-80 ml/kg/min,调节氧合器气流量保持PaCO₂介于35与45 mmHg间。24 h后各组动物停止治疗、饲养至168 h。基础状态、ALI出现时及ALI后第1天内每1h监测一次血气、Cdyn、全身血流动力学、机械通气压力、流量、通气频率、ECMO转流参数;留取血标本检测血常规、全血高铁血红蛋白及血浆亚硝酸根/硝酸根、IL-8、IL-6含量检查。168 h处死动物,行一侧肺灌洗、另一侧肺灌流固定和组织形态学检查。支气管肺泡灌洗液（bronchoalveolar lavage fluid,BALF）中白细胞计数（white cell count,WCC）、总蛋白（total proteins,TP）、总磷脂（total phospholipids,TPL）和饱和磷脂（disaturated phosphatidylcholine,DSPC）、总磷脂表面张力及亚硝酸根/硝酸根、IL-6、IL-8含量测定;肺组织湿/干重比（wet-to-dry lung weight ratio,W/D）;肺病理形态学检查,对肺泡扩张、肺损伤特点评分。肺组织中分析测定髓过氧化物酶（myeloperoxidase,MPO）、丙二醛（malondialdehyde,MDA）、谷胱甘肽（glutathione,GSH）、总NOS（total nitric oxide synthase,tNOS）活性、亚硝酸根/硝酸根含量（nitrite/nitrate,NO₂^-/NO₃^-）;IL-8、IL-6、角化细胞生长因子（keratinocyte growth factor,KGF）、肝细胞生长因子（hepatocyte growth factor,HGF）、血管内皮生长因子（vascular endothelial grow factor,VEGF）、血管内皮生长因子受体（vascular endothelial grow factor receptor,VEGFR）-2、胶原（Collagen）Ⅲ、诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）、内皮型一氧化氮合酶（endothelial nitric oxide synthase,eNOS）mRNA的表达,判断肺组织细胞炎症反应、致损伤程度及修复状况。结果:1.ALI模型的建立:在内毒素输入0.5-1 h后幼猪出现皮肤-过性紫斑、反应差、体温升高、血压下降、心率增快、呼吸急促、呼吸系统动态顺应性进行性下降、动脉血氧分压降低,同时伴有外周血白细胞数量降低。ALI在输注LPS 4-8 h内可制备成功。肺内为双肺间质弥漫性炎性细胞浸润、肺泡萎陷、出血等病理表现。2.治疗期间血流动力学:治疗期间,ECMO组、ENOS组血流动力学稳定; ECMO组平均动脉压（mean arterial blood pressure,MABP）、左心室收缩力指数（index of left ventricular contractility,dp/dtmax）、外周血管阻力（systemic vascular resistance,SVR）水平分别高于VENT组19%、25%、36%。3.治疗期间呼吸力学:ECMO、ENOS组通气频率（repiratory rate,RR）呈下降趋势;8 h、16 hECMO组RR显著低于VENT、VENOS组（p<0.01）。24 hECMO组RR显著低于VENOS组（p<0.05）。ECMO组8 h、16 h分钟通气量（minute ventilation volume,MV）显著低于VENT、VENOS组（p<0.01）;ENOS组8 h MV显著低于VENT组（p<0.05）。4.治疗期间气体交换:ECMO、ENOS组PaO₂/FiO₂呈持续、显著的改善;VENT、VENOS组PaO₂/FiO₂比值升高。5.BALF磷脂含量、表面张力、WCC和TP检测:ENOS、NENOS组DSPC显著高于VENT、VENOS组（p<0.05）;ECMO组DSPC显著高于VENT组（p<0.05）。ENOS、NENOS组最小表面张力(minimum surface tension,γ_min)显著低于VENT组（p<0.05）。ENOS、NENOS组BALF WCC显著低于VENT组（p<0.05）。各组间TP无显著性差异。6.NO₂^-/NO₃^-（NOx^-）检测:0 h,VENT、ECMO组血浆NOx^-含量显著高于NENOS组含量（p<0.05）;4 h,VENT组血浆NOx^-含量显著高于ENOS、NENOS组含量（p<0.05）。4 h后各组血浆均降低,组间无显著性差异。VENT组BALF内NOx^-含量显著高于VENOS、ECMO、ENOS、NENOS组含量（p< 0.01）见图1。7.IL-6检测:实验中各组血浆IL-6水平逐渐升高,24 h达峰值,24 h后均降低。血浆、BALF IL-6水平各组间无显著差异。8.IL-8检测:实验中血浆IL-8水平呈上升趋势,24 h达峰值。16 h,VENT组血浆IL-8含量显著高于ENOS、NENOS组含量（P<0.05）;24 h,VENT、VENOS、ECMO组血浆IL-8含量均显著高于ENOS、NENOS组含量（P< 0.05）。见图2。BALF内IL-8含量各组间差异无统计学意义。9.肺组织IL-6、IL-8、iNOS、eNOS mRNA表达:肺组织IL-6、eNOS各组间表达无显著差异。VENT组IL-8表达显著高于VENOS组、ENOS组和NENOS组（P<0.01）;ECMO组IL-8表达显著高于ENOS组和NENOS组（P<0.05）见图3。VENT组iNOS表达显著高于ECMO、ENOS、NENOS组（P<0.01）见图4。10.肺组织VEGF、VEGFR-2、KGF、HGF、collagenⅢmRNA表达:肺组织VEGF、KGF、collagenⅢ各组间表达无显著差异。NENOS组VEGFR-2表达显著高于VENT组（P<0.05）。ENOS、NENOS组HGF表达显著高于VENT组（P<0.05）。11.肺组织MPO、MDA、GSH、tNOS、NOx^-、W/D检测:各组间MPO、GSH、tNOS、NOx^-无显著差异。ENCP、NENOS组MDA含量显著低于VENT组（P<0.05）。NENOS组W/D显著低于VENT组（P<0.05）。12.肺组织学检查:VENT组动物肺有显著的粒细胞浸润、肺泡萎陷。VENOS、ECMO组存在中度病理改变。ENOS、NENOS组动物肺内有轻度粒细胞浸润。ENOS、NENOS组肺扩张度显著高于VENT组（P<0.05）。结论:1.成功制备内毒素诱导幼猪ALI模型,为开展ECMO治疗研究及观察恢复期肺组织的修复机制提供合适方法。2.ECMO治疗启动幼猪全身炎症反应并引起肺损伤。3.ECMO治疗能改善急性期ALI幼猪呼吸力学、氧合、血流动力学状态,恢复期通过降低肺内iNOS表达、NO合成减轻呼吸机治疗诱导的炎症反应。4.联用iNO、Surf通过降低急性期血浆IL-8合成、恢复期肺内IL-8表达减轻ECMO诱导的炎症反应,并可能降低呼吸机、ECMO治疗后肺MDA的合成量。通过上述机制,联用iNO、Surf上调HGF的表达,进而促进ECMO治疗后ALI幼猪肺内AEC的增殖与修复。更多还原

【Abstract】 BackgroundAcute lung injury （ALI） and acute respiratory distress syndrome （ARDS） are the most severe forms of acute and persistent hypoxemic respiratory failure （PHRF） in adults and children. ALI and ARDS in children have very high mortality and morbidity in recent domestic multicenter clinical investigations. Pathogenesis and phathophysiology of ALI/ARDS involve variable insults as pulmonary or extra-pulmonary origin, and severe alveolar-to-vascular permeability, leading to bilateral infiltration, edema, intra-pulmonary shunting and ventilation-perfusion mismatching. Clinically it is characterized as refractory hypoxaemia requiring aggressive ventilation and intensive care to survive. Currently, its death rate is more than 50% in Chinese pediatric intensive care unit （PICU）. Although various interventions with lung protective ventilation strategy are implemented and tend to be effective, no any single therapy claims cost-effective in pediatric ALI/ARDS yet. Very often, combined or alternative therapies such as lung tidal volume restriction, and alveolar recruitment including high frequency oscillation （HFOV）, fluid restriction, prone position, inhaled nitric oxide （iNO）, and exogenous surfactant （Surf）, are considered appropriate. These treatment modalities depend on effective gas exchange and adequate pulmonary perfusion to improve oxygenation. When there is dysfunction at any level of ventilation and perfusion due to sever injury in the lungs, PHRF and ALI occur. As ALI is early phase of ARDS, it is obvious that early intervention with effective and adequate therapy is vital in bringing up optimal response and outcome prediction in the very sick children.Extracorporeal membrane oxygenation （ECMO） is a unique therapy for life support in those who have impaired respiratory and circulatory function. It improves oxygenation with minimum ventilation requirement, thus enabling lung rest for reparation. Technology of ECMO involves an extracorporeal circuit conducting deoxygenated venous blood flow, after re-oxygenation and warming, to systemic circulation, either through artery or vein depending on whethere there is a heart or lung failure. It removes CO₂ out of, while fresh O₂ is provided to, the circulation, through convection of gas and blood flow in the oxygenator, or as an artificial lung. ECMO treatment also has adverse effects as it provokes a systemic inflammatory response as reflected by neutropenia, activation of polymorphonuclearcytes, release of proinflammatory cytokines such as tumor necrosis factor （TNF）-α, interleukin （IL）-1β, IL-6, IL-8, activation of complement, and a capillary leak syndrome with systemic and pulmonary edema, in addition to hemolysis of damaged red blood cell in the circuit. This may lead to secondary organ injury. Therefore we consider a modulation or down-regulation of the inflammatory process in the lungs during ECMO should alleviate lung injury and improve the prognosis of children with PHRF and ALL In our previous, as well as others, studies, iNO and/or Surf tend to be effective in improving oxygenation and inhibiting pulmonary inflammation in experimental ALI/ARDS induced by intravenous endotoxin, oleic acid, or by intra-tracheal or intra-abdominal bacteria. iNO is capable of selectively dilating intrapulmonary resistant vessels, reducing pulmonary artery hypertension, improving hypoxemia by reducing intrapulmonary shunt, and optimizing ventilation-perfusion matching. It also reveals anti-inflammatory capability by inactivating nuclear transcription factor （NF）-κΒand its downstream proinflammatory cytokine synthesis. iNO may also decrease the expression of adhesion molecules, preventing neutrophil adhesion and migration in the injured lungs. Pulmonary surfactant, a mixture of phospholipids and specific proteins produced by the typeⅡalveolar epithelial cells, is important in maintaining alveolar expansion during breath. The rationale for the use of exogenous surfactant in the treatment of patients with ALI/ARDS is not only to recover the function of surfactant, but also to inhibit the stimulated production of superoxide anions, to suppress the stimulated secretion and synthesis of proinflammatory cytokines, such as TNF-α, IL-6, IL-8, and also to inhibit granulocyte activation. Previous studies from this lab have demonstrated that a combined administration of iNO and Surf has better therapeutic effects than either therapy alone. It is of interest to know whether this combined use may exert similar effects of anti-inflammation in ECMO, with special emphasis on endotoxin-induced ALI, and any benefit in facilitating lung repair during recovery from ECMO. Objectives1. To observe ECMO effects on lung mechanics, gas exchange, and hemodynamics and the inflammation induced by ECMO in ALI piglets between 0 and 24 h, and to detect the lung pathology and biochemical injury in piglets survived at 168 h.2. To investigate the effects of a combined use of iNO and surfactant on mitigation of the lung inflammatory injury induced by ECMO in healthy and ALI piglets, and to observe the effects on lung reparation in the survived animals.MethodsAfter sedated intramuscularly with ketamine, twenty-three piglets, male, 4-5 week-old, body weight 9-14 kg, received i.v. infusion of LPS （18-20μg/kg） within one h, followed by mechanical ventilation with a standard tidal volume of 7-9 ml/kg for 4-8 h. ALI was defined as PaO₂/FiO₂≤300 mmHg, dynamic lung compliance （Cdyn） decreased by more than 30% of its baseline level. This moment was regarded as treatment time 0 h. At 0, 12, and 24 h three animals were immediately sacrificed. Their lungs showed that LPS induced diffuse alveolar damage represented by alveolar atelectasis, and leukocyte sequestration. The other 20 ALI animals were randomly allocated to four groups （n=5） and defined as: VENT group, animals treated with PCV ventilation; VENOS group, animals treated with PCV ventilation, inhalation of 10 ppm NO, 50 mg of surfactant phospholipids/kg body weight via the endotracheal tube; ECMO group, animals treated with PCV ventilation, ECMO; ENOS group, animals treated with PCV ventilation, ECMO, iNO and surfactant as VENOS group. Additionally, five healthy piglets were used as a normal control group （NENOS group）. Animals in NENOS group were treated with PCV ventilation, ECMO, iNO and surfactant. During the experiment, Ringer’s lactate solution was i.v. infused to keep normal blood pressure, and 1.4% bicarbonate sodium in Ringer’s solution was given to overcome metabolic acidosis. FiO₂ was adjusted to maintain PaO₂ greater than 60 mm Hg, and PaCO₂ was maintained between 35 and 45 mm Hg by varying the respiratory rate （RR） in the non-ECMO-treated animals. Continuous infusion of heparin maintained the activated clotting time （ACT） at 180 to 220 s in ECMO groups. The ECMO flow was kept 70-80 ml/kg/min. The initial sweep gas flow of oxygenator was set at 2 L/min and titrated to keep PaCO₂ between 35 to 45 mm Hg. Animals were treated with above settings from 0 to 24 h and fed between 24 h and 168 h. Arterial blood gas, Cdyn, systematic hemodynamics, airway pressure, minute ventilation volume, RR of ventilator, and ECMO parameters were monitored at the baseline, establishment of ALI, and each h during the treatment. Blood samples were collected at baseline, establishment of ALI, 4, 8, 16, 24, 48, and 168 h of the treatment. At 168 h, animals were sacrificed by overdose of 10% potassium chloride and lung tissues and bronchoalveolar lavage fluid （BALF） were collected. Total proteins （TP）, total phospholipids （TPL）, disaturated phosphatidylcholine （DSPC） were measured with biochemical methods and minimum and maximum surface tension (γ_min andγ_max) of TPL in BALF were measured using pulsating bubble technique. Commercial available kits were used to measure the levels of NOx^-, IL-8, IL-6, myeloperoxidase （MPO） malondialdehyde （MDA）, glutathione （GSH）, and total nitric oxide synthase activity （tNOS）. The expression of IL-8, IL-6, keratinocyte growth factor （KGF）, hepatocyte growth factor （HGF）, vascular endothelial grow factor （VEGF）, vascular endothelial grow factor receptor 2 （VEGFR-2）, collagenⅢ, inducible nitric oxide synthase （iNOS）, and endothelial nitric oxide synthase （eNOS） mRNA in lung tissues were measured by real-time polymerase chain reaction （real-time PCR） at the end of the experiment.Results1. ALI model: Piglets appeared transient purple plague, poor response, the increase of blood temperature and heart rate, tachypnea, and the decrease of systemic blood pressure, Cdyn and PaO₂ and accompanied with the decrease of peripheral WBC 0.5-1 hour since LPS infused. It took 4-8 h to result in ALI. The lung pathology showed that LPS induced diffuse alveolar damage represented by alveolar bleeding, atelectasis, leukocyte sequestration.2. Hemodynamics during treatment: During the treatment, the systemic hemodynamics of ECMO and ENOS groups was stable. Levels of MABP, dp/dtmax, and SVR in ECMO group were 19%, 25%, and 36% higher than those in VENT group respectively.3. Lung mechanics during treatment: There was a reducing trend of RR in the ECMO and ENOS groups. At 8 and 16 h, RR of ECMO group was significantly lower than that of VENT and VENOS groups （p<0.01）. At 24 h, RR of VENOS was obviously higher in comparison to that of ECMO group （p<0.05）. At 8 and 16 h, minute ventilation volume （MV） of ECMO group was significantly lower than that of VENT and VENOS groups （p < 0.01）. Minute ventilation volume of 8 h in ENOS group was significantly lower compared to that in VENT group （p < 0.05）.4. Gas exchange during treatment: There was a continuous and significant improvement in PaO₂/FiO₂ in ECMO and ENOS groups during ECMO treatment and it was improved in both VENT and VENOS groups as well.5. Phospholipids, surface tension, white cell counts （WCC）, and TP in BALF: ENOS and NENOS groups had higher DSPC than that of VENT and VENOS groups （p < 0.05） and had lower minimum surface tension of TPL in BALF than that of VENT group （p < 0.05）. ECMO group had higher DSPC than that of VENT （p < 0.05）. WCC in ENOS and NENOS groups was lower than that in VENT group （p < 0.05）. There was not significant difference in TP levels among the groups.6. NOx^- concentration: At 0 h, plasma NOx^- of VENT and ECMO groups was significantly higher than that of NENOS group （p < 0.05）. At 4 h, VENT group had significantly higher NOx^- than ENOS and NENOS groups （p < 0.05）. At 168 h, BALF NOx^- of VENT group was higher than that of VENOS, ECMO, and NENOS groups （p< 0.01）.7. IL-6 concentration: During the treatment, plasma IL-6 in all groups increased and reached its peak level at 24 h. At 48 and 168 h, this level was decreased in all groups.8. IL-8 concentration: During the treatment, plasma IL-8 in all groups increased and reached its peak level at 24 h. At 16 h, plasma IL-8 of VENT group was significantly higher than that in ENOS and NENOS （p < 0.05）. At 24 h, VENT, VENOS, and ECMO had higher plasma IL-8 than that in ENOS and NENOS （p < 0.05）. There was not significant difference in BALF IL-8 among the groups.9. The mRNA expression of IL-6, IL-8, iNOS, and eNOS in the lung tissue: There were no significant differences in mRNA expression of IL-6 and eNOS among the groups. IL-8 expression in VENT was significantly higher than that in VENOS, ENOS, and NENOS （p < 0.01）. ECMO had higher IL-8 expression than that in ENOS and NENOS （p < 0.05）. VENT had significantly higher iNOS expression than that in ECMO, ENOS, and NENOS （p < 0.01）.10. The mRNA expression of VEGF, VEGFR-2, KGF, HGF, and collagenⅢin lung tissues: There were no significant differences in mRNA expression of VEGF, KGF, and collagenⅢamong the groups. Expression of VEGFR-2 in NENOS was significantly higher than that in VENT （p < 0.05）. ENOS and NENOS had higher expression of HGF than in VENT （p < 0.05）.11. MPO, MDA, GSH, tNOS, NOx^-, and W/D in lung tissue: There were not significant differences in the levels of MPO, GSH, tNOS, and NOx^- in lung tissue among the groups. MDA in ENOS and NENOS was significantly lower than that in VENT （p < 0.05）, and VENT group had higher W/D than that in NENOS group （p < 0.05）.12. Lung histopathology: There was prominent neutrophil infiltration in VENT. VENOS and ECMO had moderate pathological changes. There was modest neutrophil infiltration in ENOS and NENOS. Volume density of alveolar aeration in ENOS and NENOS was significantly higher than in VENT （p < 0.05）.Conclusions1. ALI was successful established by intravenous administration of LPS in young piglets, which enabled assessment of therapeutic efficacy and safety of ECMO and investigation of mechanicsm of lung injury and repair in the recovery.2. ECMO initiated a systemic inflammatory response and caused lung injury in these animals.3. ECMO improved lung mechanics, oxygenation, and hemodynamic condition of ALI piglets in acute phase and alleviated the lung inflammatory response induced by ventilator treatment associated with differential expression of iNOS and endogenous NO metabolites in the lungs.4. Combined use of iNO and surfactant mitigated the inflammatory response provoked by ECMO as reflected by plasma IL-8 production in the acute phase and the lung expression of IL-8 in the recovery phase as well as altered MDA production. This modality upgraded the expression of HGF and facilitated reparation of alveolar epithelial cells in the recovery phase.更多还原