VEGF和Ang在新生鼠高氧肺损伤的表达及相关机制研究
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摘要
研究背景
支气管肺发育不良(Bronchopulmonary dysplasia,BPD)是早产儿生后由于急性呼吸窘迫长期应用高浓度氧气和呼吸机辅助通气治疗后的一种慢性肺部疾病,常常引起严重并发症,导致患儿长期依赖氧气,存活患儿常伴随有慢性肺功能障碍和气体交换障碍,易发生呼吸道感染、精神与运动发育落后及喂养困难等,后期可出现运动障碍、脑瘫、听力丧失等残疾,严重影响患儿生存质量。近年来遗传生殖工程技术迅速发展,试管婴儿、多胞胎比例明显增加,以及环境因素及其他多种原因,随之不可避免的早产儿尤其是超早产儿明显增多,临床上机械通气和肺表面活性物质的广泛应用,早产儿救治水平和管理技术的日益提高,目前极低体重儿和超低出生体重儿的存活率有了显著改善,随之而来的幸存者常见肺部并发症BPD的发病率必然呈现逐渐上升的趋势,从而BPD成为新生儿重症监护病房(Neonatal Intensive Care Unit, NICU)所面临的最常见而又最为棘手的问题之一,也是婴幼儿时期最常见的慢性呼吸系统疾病,严重影响早产儿的存活率和生活质量。由于BPD的定义和诊断标准方面没有完全统一,国内外对BPD的发病率报道不一。目前BPD多见于极不成熟早产儿。Stevenson报道在体重不足1000g的早产儿中,其发病率高达30%。因此,目前BPD的早期诊治和预防已成为临床上一个十分重要的课题,其早期干预及治疗意义重大。鉴于BPD患儿有较高的入院率与死亡率,其确切致病因素及致病机理尚未完全明了,故而其发病机制、预防和治疗方法的研究一直是新生儿医学和呼吸病学的热点。但目前BPD尚缺乏有效的预防措施,有部分患儿治疗效果不佳,长期不能脱氧,常因反复感染而死亡,后期生活质量不高,因此对此进行研究,探索有效的预防和治疗措施,寻找有效的早期干预及治疗途径意义重大,将有助于减少儿童致残率,提高患儿存活率和生活质量。
研究发现机械通气和高浓度氧气可使早产狒狒和羊的肺泡面积明显减少,肺血管出现形态异常改变,严重减少肺泡数量,阻碍肺泡和肺血管的发育。且早产儿肺泡化未完成,肺表面活性物质(pulmonary surfactant, PS)系统和内源性抗过氧化酶(过氧化歧化酶、谷胱甘肽过氧化酶、催化酶等)系统发育不成熟,其活性相对缺陷,更容易导致过氧化损害,使用高浓度氧气的BPD早产儿可出现更持续的肺部疾病,使囊泡阶段的肺发育停滞。同时高氧肺损伤后肺部白细胞、巨噬细胞渗出增加,许多的促炎症细胞因子(白细胞介素-1、6、8,肿瘤坏死因子a,血小板活化因子等)和趋炎因子释放,炎症形成了BPD的中心,限制了出生后肺泡、小呼吸道和小血管的发育,抑制出生后的肺泡化,从而导致肺发育停滞。
BPD的发病机制较为复杂,其确切致病因素及致病机理目前尚未完全明了,其高危致病因素主要包括：长时间高浓度吸氧导致氧中毒,呼吸机辅助机械通气引起的气压伤和容量伤,早产儿肺发育的不成熟,感染炎症所导致的肺损伤,以及遗传因素等。其中肺发育的不成熟和氧中毒是最关键的致病因素。在医学界已经得到普遍认同,吸入高浓度氧是引起早产儿BPD的主要原因之一。国内外研究认为肺发育停滞是其主要的病理改变。既往BPD的发病机制的报道多着重于肺泡化障碍的机制研究,然而肺发育包括肺泡发育和血管发育两个方面,肺发育阶段肺泡分化的同时微血管网同步扩展,形成有效的气血交换屏障。越来越多的证据表明肺血管在发育过程中主动促进肺泡的正常生长,并且在生后参与对肺泡结构的维持。血管内皮生长因子(Vascular endothelial growth factor,VEGF)和血管生成素(Angiopoietin, Ang)是肺发育过程中肺血管发育最重要的两种因子。
VEGF是一种多功能的细胞因子,是整个胚胎期、胎儿期和生后肺血管生长与维持的最有力和最关键的调节因子,并参与了新生儿高氧肺损伤的发生。Ang是特异性作用于血管内皮的血管生长因子。VEGF主要作用于肺血管形成的早期,促进原始血管网的形成；Ang-1作用于肺血管重建、塑形,促进成熟而有空间结构的血管网的形成。动物实验表明VEGF和Ang可能具有协同作用。Ang-2可能通过抑制Ang-1来增强内皮细胞对VEGF的反应。Ang-2可拮抗Ang-1促血管稳定的作用,消除血管基底膜和管周细胞对血管形成的限制,并增加内皮细胞对VEGF的敏感性,有利于血管出芽、生长；血管内皮细胞上Ang-2可以通过抑制Ang-1活化程度,形成内分泌调节环路,来维持血管生长、退化的动态平衡,而且Ang-2的表达也受VEGF的调节。Ang还与内皮细胞凋亡关系密切。研究表明,Ang-1可抑制人脐带血管内皮细胞的凋亡且存在剂量依赖关系,并与VEGF有协同效应。目前研究证明Ang-3也受到VEGF的调控,Motoi等研究显示VEGF可上调人脐静脉内皮细胞中Ang-3的表达,并且下调Ang-2的表达。Ang-1还是一种很好的抗炎因子。Gamble等研究显示,Ang-1能加强内皮细胞与细胞外基质,管周细胞间的连接,维持血管内皮细胞单层紧密排列的形态,有效防止血浆外漏,Ang-1还能抑制由VEGF诱发血管渗漏导致的组织水肿。Ang和VEGF间的相互作用对血管成熟是很重要的,但它们间的相互作用很复杂并且与许多因素有关,目前尚不明确。研究VEGF、Ang在高氧诱导新生鼠肺损伤时的动态表达及其对肺发育影响为支气管发育不良的相关机制研究及改善肺发育障碍等方面奠定了实验基础。
实时荧光定量PCR是近年发展起来的一项新的核酸检测技术。其原理是在PCR反应体系中加入荧光基团,利用荧光信号积累实时监测整个PCR进程,最后通过标准曲线对未知模板进行定量分析的方法。该技术不仅实现了PCR从定性到定量的飞跃,而且与常规PCR相比,它具有特异性更强、有效解决PCR污染问题、自动化程度高等特点,目前已得到广泛应用。免疫印迹(immunoblotting)又称为蛋白质印迹(Western blotting),是在凝胶电泳和固相免疫测定技术基础上发展起来的一种新的免疫生化技术,是根据抗原抗体的特异性结合检测复杂样品中的某种蛋白的方法。该法由于免疫印迹具有SDS-PAGE的高分辨力和固相免疫测定的高特异性和敏感性,现已成为蛋白分析的一种常规技术。采用实时荧光定量PCR技术和蛋白质免疫印迹技术检测高氧诱导新生SD大鼠肺损伤时肺组织中VEGF和Ang基因和蛋白的动态表达水平,有助于进一步证实高氧肺损伤时VEGF和Ang在肺损伤中的作用及其对肺发育的影响。
合适的实验动物模型是研究BPD的关键。大型灵长类动物如早产狒狒和羊其肺发育与人类相似,且可以进行呼吸机辅助机械通气,是有价值的实验研究动物,但由于价格昂贵,需要较高技术的监护设备,实施起来存在较大困难。而小型啃噬类动物如大鼠、小鼠等由于其价格方面和管理方面的优势已被广泛用于高氧肺损伤及BPD的实验动物模型的研究。研究显示新生SD大鼠出生后2-3天日龄相当于人类新生儿期。已有文献对亚致死浓度(<85%)和中等氧浓度(50%～65%)对肺发育阻滞和高氧肺损伤进行研究,但实验所需时间较长,多为2～4周,而致死浓度(>90%)更容易造成肺损伤,更符合研究需要。95%高浓度氧是研究动物高氧肺损伤常用的浓度,短期可造成肺急性炎性改变,延长给氧时间则可能导致肺形态结构变化。本研究动物实验的预实验结果显示本研究建立高氧诱导新生Sprague-Dawley大鼠肺损伤模型的预实验结果显示,高氧组第7天肺组织表现类似BPD的病理改变。故本课题采用95%高浓度氧气暴露诱导2-3天日龄的新生SD大鼠肺损伤建立BPD实验动物模型。
研究目的
1.通过建立高氧损伤诱导的新生鼠BPD模型,观察高氧暴露后的新生大鼠肺组
织形态学改变情况,从肺血管发育的角度进一步揭示BPD发生的病理机制；
2.探讨VEGF、Ang在肺血管发育中的动态表达以及与高氧诱导肺损伤致支气管
肺发育不良的关系,为完善早产儿BPD的发生机制及寻求有效的防治途径提供实验依据,以期应用于临床实验研究,从而改善BPD患儿的预后,提高早产儿的存活率和生活质量。
研究内容和方法
1.首先建立新生鼠高氧肺损伤的实验动物模型：以新生2-3天日龄SD大鼠为研究对象,雌雄不限,高浓度氧(≥95%)暴露1天、3天和7天,并建立BPD高氧肺损伤的动物模型(高浓度氧≥95%7天)；
2.将新生SD大鼠正常对照组和高氧损伤组平均分为3个小组；高氧损伤组分别高浓度氧(≥95%)暴露1天、3天和7天,利用光学显微镜动态观察各组肺组织形态学变化；
3.设计引物和内参照,用实时荧光定量PCR (RT-PCR)方法和蛋白质免疫印迹(Western Blotting)半定量检测各时点样本VEGF、Ang mRNA和蛋白的表达；结合肺组织形态学变化,评价高氧肺损伤时VEGF、Ang表达及其对肺发育的影响。
结果
1.新生SD大鼠高氧肺损伤BPD模型的构建：新生2-3天SD大鼠持续≥95%的高氧暴露7天,表现为活动减少,反应迟钝,体重和身长均增长缓慢,高氧损伤组与正常对照组比较,差异具有统计学意义(P<0.05)。
2.光学显微镜动态观察各组肺组织形态学变化：低倍光镜下观察正常对照组新生鼠肺组织未见病理改变,表现为肺泡结构清楚、大小均一,无液体和炎性渗出；高氧损伤组第1天、第3天和第7天肺组织形态逐渐出现肺泡结构简单化、肺泡数目明显减少,肺泡大小不均,肺泡壁较薄,有些肺泡融合、体积增加；高倍镜观察,正常对照组肺泡壁完整,肺泡内未见明显脱落细胞；高氧损伤组新生鼠肺泡中见有红细胞和巨噬细胞渗出,并可见脱落的肺上皮细胞。高氧损伤组与正常对照组比较逐渐表现出肺泡数目减少,直径变大,肺泡面积减小,肺泡间隔厚度减小,差异具有统计学意义(P<0.05)。
3.正常对照组第1天、第3天和第7天VEGF mRNA和蛋白的表达：正常对照组第1天、第3天和第7天VEGF mRNA相对含量分别为：1.000±0.366、0.931±0.370、0.722±0.372；正常对照组第1天、第3天和第7天VEGF蛋白相对含量分别为：1.000±0.498、0.787+0.544、0.632±0.289；正常组第1天、第3天和第7天VEGF mRNA和蛋白相对含量比较,均无显著性差异(P>0.05)。
4.正常对照组第1天、第3天和第7天Ang mRNA和蛋白的表达：正常对照组第1天、第3天和第7天Ang mRNA相对含量分别为：1.000-±1.108、0.907±1.019、0.828±0.462;正常对照组第1天、第3天和第7天Ang蛋白相对含量分别为：1.000-±0.858、0.995±0.871、0.573±0.436；正常组第1天、第3天和第7天Ang mRNA和蛋白相对含量比较,均无显著性差异(P>0.05)。
5.高氧损伤组第1天、第3天和第7天VEGF mRNA和蛋白的表达：高氧损伤组第1天、第3天和第7天VEGF mRNA相对含量分别为：0.985±0.403、0.897±0.512、0.239±0.293；高氧损伤组第1天、第3天和第7天VEGF蛋白相对含量分别为：0.960±0.619、0.730±0.342、0.358±0.128；高氧损伤组第7天新生鼠肺组织VEGF mRNA和蛋白表达水平均明显低于正常对照组第7天,存在显著差异(P<0.05)。
6.高氧损伤组第1天、第3天和第7天天Ang mRNA和蛋白的表达：高氧损伤组第1天、第3天和第7天Ang mRNA相对含量分别为：1.011±1.131、0.843±0.388、0.327±0.184；高氧损伤组第1天、第3天和第7天Ang蛋白相对含量分别为：0.755±0.414、0.711±0.539、0.204±0.068；高氧损伤组第7天新生鼠肺组织Ang mRNA和蛋白表达水平均明显低于正常对照组第7天,存在显著差异(P<0.05)。
结论
1.通过新生2-3天日龄SD大鼠持续吸入高浓度氧气(≥95%)7天可构建BPD高氧肺损伤的动物模型。
2.正常新生SD大鼠肺组织中VEGF、Ang表达呈动态变化趋势,提示VEGF、Ang在肺组织的动态表达与肺发育之间有着密切关系,进一步说明VEGF、Ang参与了肺发育过程。
3.高氧可诱导支气管肺发育不良的发生,随着高氧暴露时间的延长,高氧肺损伤时VEGF、Ang mRNA和蛋白表达均明显减少,光镜下肺组织形态逐渐呈现高氧肺损伤时肺泡发育停滞的肺部病理改变,提示高氧肺损伤时VEGF、Ang表达减少可能通过阻碍肺发育参与BPD模型鼠高氧肺损伤的发生机制,为BPD的早期干预奠定实验基础。
Bronchopulmonary dysplasia (BPD) is an important complication of premature infants, and it is a common chronic diseases of respiratory system for infants. But its certain etiological factors and pathogenesis are not clear completely, inhaling hyperoxia for a long time to lead to lung injury is a main reason, and some investigations showed that arrested lung development was the main pathological change. As well as known that immature lung development and oxygen toxicity are the key risk factors. Inhaling hyperoxia is one of the leading causes for prmature babies' bronchopulmonary dysplasia. Normal vascular development of lung includes vasculogenesis and angiogenesis. Recently, some researches considered that abnormal expression of lung development regulating factors would influence the normal morphological and vascular development of lung. Vascular endothelial growth factor(VEGF) and Angiopoietin(Ang) are two main factors which play an important role in morphological development of lung, tissue moulding and vascular remodeling. At present, it is considered that inhaling hyperoxia leading to abnormal expression of VEGF and Ang is significant in arrested lung development. Explore related pathological mechanism of VEGF and Ang in model rats with lung injury caused by hypoxia and their influence of alveoli and vascular development of lung will provide a new strategy and direction in prevention and cure of neonatal bronchopulmonary dysplasia.
Objectives
1. Make a neonatal rats BPD model by hypoxia, to observe the pathological change of lung, reveal the pathological mechanism of BPD in further to provide experimental fundation for new ways about early intervention and gene intervention therapy.
2. Explore expression characters of VEGF and Ang mRNA and protein in neonatal rats'lungs with lung injury by hyperoxia, invesigate the relationship between them and bronchopulmonary dysplasia to provide experimental fundation for research about mechanism of BPD.
Methods
1. The neonatal Sprague-Dawley rats were used as study objects after birth for2-3days, and exposured to hyperoxia(≥95%) for1,3or7days to make a model with BPD.
2. The neonatal rats were divided into contol groups and hyperoxia groups at random; lung histological changes of neonatal Sprague-Dawley rats in each group were observed and compared through light microscope. Number, diameter, area of alveoli and alveolar septum thickness were measured by image manipulation and analysis software. Quantitive analysis of histological changes were did for comparing difference of lung histomophology appearnce between groups.
3. Expression of VEGF and Ang mRNA were detected by real time fluorescent quantitation polymerase chain reaction, and the interference effect of VEGF and Ang for lung development were evaluated combined with the morphological changes of lung.
4. Protein expression of VEGF and Ang were detected by Western blotting, and the interference effect of VEGF and Ang for lung development were evaluated combined with the morphological changes of lung.
Results
1. Make a model with lung injury, BPD of neonatal rats:neonatal rats were exposed to hyperoxia(≥95%) for7days, without activity, dull reaction, their weight and length increased slowly; compared rats' weight and length of model group with control group, it was very different (P<0.05).
2. Observation of lung histological changes of rats in each group with light microscope:in low view, the lung tissue in noraml rats showed clear and alveolar structure, uniform size, no fluid exudation; the lung tissue of hyperoxia groups displayed thinner walls of alveoli, simple alveolar structure, fewer and larger alveoli, expanded and shrinked alveoli, decreased area of alveoli. In high sight, the lung tissue in normal rats showed complete walls of alveoli, no cells in alveoli; the lung tissue of model groups with hyperoxia showed few red cells, macrophages and AECⅡ in space of alveoli. Compare area, diameter of alveoli and thickness of alveolar septum of model groups with control groups, it was very different (P<0.05).
3. VEGF mRNA and protein expressions of control groups detected by real time fluorescent quantitation polymerase chain reaction and Western blotting:VEGF mRNA relative expression in control groups were1.000±0.366,0.931±0.370or0.722±0.372; VEGF protein relative expression in control groups were1.000±0.498,0.787±0.544or0.632±0.289; compared VEGF mRNA and protein expressions between them, it wasn't different (P>0.05).
4. Ang mRNA and protein expressions of control groups detected by real time fluorescent quantitation polymerase chain reaction and Western blotting:Ang mRNA relative expression in control groups were1.000±1.108,0.907±1.019or 0.828±0.462; Ang protein relative expression in control groups were1.000±0.858,0.995±0.871or0.573±0.436; compared Ang mRNA and protein expressions between them, it wasn't different (P>0.05).
5. VEGF mRNA and protein expressions of hyperoxia groups detected by real time fluorescent quantitation polymerase chain reaction and Western blotting:VEGF mRNA relative expression in control groups were0.985±0.403,0.897±0.512or0.239±0.293; VEGF protein relative expression in control groups were0.960±0.619,0.730±0.342or0.358±0.128; compared VEGF mRNA and protein expressions of the7th days between control group and hyperoxia group, it was very different (P<0.05).
6. Ang mRNA and protein expressions of hyperoxia groups detected by real time fluorescent quantitation polymerase chain reaction and Western blotting:Ang mRNA relative expression in control groups were1.011±1.131,0.843±0.388or0.327±0.184; Ang protein relative expression in control groups were0.755±0.414,0.711±0.539or0.204±0.068; compared Ang mRNA and protein expressions of the7th days between control group and hyperoxia group, it was very different (P<0.05).
Conclusions
1. Inhaling hyperoxia(≥95%) for7days persistantly could induced lung injury leading to a model of BPD.
2. VEGF and Ang mRNA and protein expression decreased with lung injury by hyperoxia, which showed that VEGF and Ang might take important part in the pathological mechanism of model rats with BPD by hyperoxia through promoting alveoli and vascular development of lung.
3. Inhaling hyperoxia persistantly could induced lung injury leading to a model of BPD, and the lung tissue of hyperoxia groups displayed thinner walls of alveoli, simple alveolar structure, fewer and larger alveoli, expanded and shrinked alveoli, decreased area of alveoli, which showed that VEGF and Ang might take an important part in lung development from lung injure of hyperoxia, and they mighty cooperate or restrict with each other..
支气管肺发育不良(Bronchopulmonary dysplasia,BPD)是早产儿生后由于急性呼吸窘迫长期应用高浓度氧气和呼吸机辅助通气治疗后的一种慢性肺部疾病,常常引起严重并发症,导致患儿长期依赖氧气,存活患儿常伴随有慢性肺功能障碍和气体交换障碍,易发生呼吸道感染、精神与运动发育落后及喂养困难等,后期可出现运动障碍、脑瘫、听力丧失等残疾,严重影响患儿生存质量。近年来遗传生殖工程技术迅速发展,试管婴儿、多胞胎比例明显增加,以及环境因素及其他多种原因,随之不可避免的早产儿尤其是超早产儿明显增多,临床上机械通气和肺表面活性物质的广泛应用,早产儿救治水平和管理技术的日益提高,目前极低体重儿和超低出生体重儿的存活率有了显著改善,随之而来的幸存者常见肺部并发症BPD的发病率必然呈现逐渐上升的趋势,从而BPD成为新生儿重症监护病房(Neonatal Intensive Care Unit, NICU)所面临的最常见而又最为棘手的问题之一,也是婴幼儿时期最常见的慢性呼吸系统疾病,严重影响早产儿的存活率和生活质量。由于BPD的定义和诊断标准方面没有完全统一,国内外对BPD的发病率报道不一。目前BPD多见于极不成熟早产儿。Stevenson报道在体重不足1000g的早产儿中,其发病率高达30%。因此,目前BPD的早期诊治和预防已成为临床上一个十分重要的课题,其早期干预及治疗意义重大。鉴于BPD患儿有较高的入院率与死亡率,其确切致病因素及致病机理尚未完全明了,故而其发病机制、预防和治疗方法的研究一直是新生儿医学和呼吸病学的热点。但目前BPD尚缺乏有效的预防措施,有部分患儿治疗效果不佳,长期不能脱氧,常因反复感染而死亡,后期生活质量不高,因此对此进行研究,探索有效的预防和治疗措施,寻找有效的早期干预及治疗途径意义重大,将有助于减少儿童致残率,提高患儿存活率和生活质量。
研究发现机械通气和高浓度氧气可使早产狒狒和羊的肺泡面积明显减少,肺血管出现形态异常改变,严重减少肺泡数量,阻碍肺泡和肺血管的发育。且早产儿肺泡化未完成,肺表面活性物质(pulmonary surfactant, PS)系统和内源性抗过氧化酶(过氧化歧化酶、谷胱甘肽过氧化酶、催化酶等)系统发育不成熟,其活性相对缺陷,更容易导致过氧化损害,使用高浓度氧气的BPD早产儿可出现更持续的肺部疾病,使囊泡阶段的肺发育停滞。同时高氧肺损伤后肺部白细胞、巨噬细胞渗出增加,许多的促炎症细胞因子(白细胞介素-1、6、8,肿瘤坏死因子a,血小板活化因子等)和趋炎因子释放,炎症形成了BPD的中心,限制了出生后肺泡、小呼吸道和小血管的发育,抑制出生后的肺泡化,从而导致肺发育停滞。
BPD的发病机制较为复杂,其确切致病因素及致病机理目前尚未完全明了,其高危致病因素主要包括：长时间高浓度吸氧导致氧中毒,呼吸机辅助机械通气引起的气压伤和容量伤,早产儿肺发育的不成熟,感染炎症所导致的肺损伤,以及遗传因素等。其中肺发育的不成熟和氧中毒是最关键的致病因素。在医学界已经得到普遍认同,吸入高浓度氧是引起早产儿BPD的主要原因之一。国内外研究认为肺发育停滞是其主要的病理改变。既往BPD的发病机制的报道多着重于肺泡化障碍的机制研究,然而肺发育包括肺泡发育和血管发育两个方面,肺发育阶段肺泡分化的同时微血管网同步扩展,形成有效的气血交换屏障。越来越多的证据表明肺血管在发育过程中主动促进肺泡的正常生长,并且在生后参与对肺泡结构的维持。血管内皮生长因子(Vascular endothelial growth factor,VEGF)和血管生成素(Angiopoietin, Ang)是肺发育过程中肺血管发育最重要的两种因子。
VEGF是一种多功能的细胞因子,是整个胚胎期、胎儿期和生后肺血管生长与维持的最有力和最关键的调节因子,并参与了新生儿高氧肺损伤的发生。Ang是特异性作用于血管内皮的血管生长因子。VEGF主要作用于肺血管形成的早期,促进原始血管网的形成；Ang-1作用于肺血管重建、塑形,促进成熟而有空间结构的血管网的形成。动物实验表明VEGF和Ang可能具有协同作用。Ang-2可能通过抑制Ang-1来增强内皮细胞对VEGF的反应。Ang-2可拮抗Ang-1促血管稳定的作用,消除血管基底膜和管周细胞对血管形成的限制,并增加内皮细胞对VEGF的敏感性,有利于血管出芽、生长；血管内皮细胞上Ang-2可以通过抑制Ang-1活化程度,形成内分泌调节环路,来维持血管生长、退化的动态平衡,而且Ang-2的表达也受VEGF的调节。Ang还与内皮细胞凋亡关系密切。研究表明,Ang-1可抑制人脐带血管内皮细胞的凋亡且存在剂量依赖关系,并与VEGF有协同效应。目前研究证明Ang-3也受到VEGF的调控,Motoi等研究显示VEGF可上调人脐静脉内皮细胞中Ang-3的表达,并且下调Ang-2的表达。Ang-1还是一种很好的抗炎因子。Gamble等研究显示,Ang-1能加强内皮细胞与细胞外基质,管周细胞间的连接,维持血管内皮细胞单层紧密排列的形态,有效防止血浆外漏,Ang-1还能抑制由VEGF诱发血管渗漏导致的组织水肿。Ang和VEGF间的相互作用对血管成熟是很重要的,但它们间的相互作用很复杂并且与许多因素有关,目前尚不明确。研究VEGF、Ang在高氧诱导新生鼠肺损伤时的动态表达及其对肺发育影响为支气管发育不良的相关机制研究及改善肺发育障碍等方面奠定了实验基础。
实时荧光定量PCR是近年发展起来的一项新的核酸检测技术。其原理是在PCR反应体系中加入荧光基团,利用荧光信号积累实时监测整个PCR进程,最后通过标准曲线对未知模板进行定量分析的方法。该技术不仅实现了PCR从定性到定量的飞跃,而且与常规PCR相比,它具有特异性更强、有效解决PCR污染问题、自动化程度高等特点,目前已得到广泛应用。免疫印迹(immunoblotting)又称为蛋白质印迹(Western blotting),是在凝胶电泳和固相免疫测定技术基础上发展起来的一种新的免疫生化技术,是根据抗原抗体的特异性结合检测复杂样品中的某种蛋白的方法。该法由于免疫印迹具有SDS-PAGE的高分辨力和固相免疫测定的高特异性和敏感性,现已成为蛋白分析的一种常规技术。采用实时荧光定量PCR技术和蛋白质免疫印迹技术检测高氧诱导新生SD大鼠肺损伤时肺组织中VEGF和Ang基因和蛋白的动态表达水平,有助于进一步证实高氧肺损伤时VEGF和Ang在肺损伤中的作用及其对肺发育的影响。
合适的实验动物模型是研究BPD的关键。大型灵长类动物如早产狒狒和羊其肺发育与人类相似,且可以进行呼吸机辅助机械通气,是有价值的实验研究动物,但由于价格昂贵,需要较高技术的监护设备,实施起来存在较大困难。而小型啃噬类动物如大鼠、小鼠等由于其价格方面和管理方面的优势已被广泛用于高氧肺损伤及BPD的实验动物模型的研究。研究显示新生SD大鼠出生后2-3天日龄相当于人类新生儿期。已有文献对亚致死浓度(<85%)和中等氧浓度(50%～65%)对肺发育阻滞和高氧肺损伤进行研究,但实验所需时间较长,多为2～4周,而致死浓度(>90%)更容易造成肺损伤,更符合研究需要。95%高浓度氧是研究动物高氧肺损伤常用的浓度,短期可造成肺急性炎性改变,延长给氧时间则可能导致肺形态结构变化。本研究动物实验的预实验结果显示本研究建立高氧诱导新生Sprague-Dawley大鼠肺损伤模型的预实验结果显示,高氧组第7天肺组织表现类似BPD的病理改变。故本课题采用95%高浓度氧气暴露诱导2-3天日龄的新生SD大鼠肺损伤建立BPD实验动物模型。
研究目的
1.通过建立高氧损伤诱导的新生鼠BPD模型,观察高氧暴露后的新生大鼠肺组
织形态学改变情况,从肺血管发育的角度进一步揭示BPD发生的病理机制；
2.探讨VEGF、Ang在肺血管发育中的动态表达以及与高氧诱导肺损伤致支气管
肺发育不良的关系,为完善早产儿BPD的发生机制及寻求有效的防治途径提供实验依据,以期应用于临床实验研究,从而改善BPD患儿的预后,提高早产儿的存活率和生活质量。
研究内容和方法
1.首先建立新生鼠高氧肺损伤的实验动物模型：以新生2-3天日龄SD大鼠为研究对象,雌雄不限,高浓度氧(≥95%)暴露1天、3天和7天,并建立BPD高氧肺损伤的动物模型(高浓度氧≥95%7天)；
2.将新生SD大鼠正常对照组和高氧损伤组平均分为3个小组；高氧损伤组分别高浓度氧(≥95%)暴露1天、3天和7天,利用光学显微镜动态观察各组肺组织形态学变化；
3.设计引物和内参照,用实时荧光定量PCR (RT-PCR)方法和蛋白质免疫印迹(Western Blotting)半定量检测各时点样本VEGF、Ang mRNA和蛋白的表达；结合肺组织形态学变化,评价高氧肺损伤时VEGF、Ang表达及其对肺发育的影响。
结果
1.新生SD大鼠高氧肺损伤BPD模型的构建：新生2-3天SD大鼠持续≥95%的高氧暴露7天,表现为活动减少,反应迟钝,体重和身长均增长缓慢,高氧损伤组与正常对照组比较,差异具有统计学意义(P<0.05)。
2.光学显微镜动态观察各组肺组织形态学变化：低倍光镜下观察正常对照组新生鼠肺组织未见病理改变,表现为肺泡结构清楚、大小均一,无液体和炎性渗出；高氧损伤组第1天、第3天和第7天肺组织形态逐渐出现肺泡结构简单化、肺泡数目明显减少,肺泡大小不均,肺泡壁较薄,有些肺泡融合、体积增加；高倍镜观察,正常对照组肺泡壁完整,肺泡内未见明显脱落细胞；高氧损伤组新生鼠肺泡中见有红细胞和巨噬细胞渗出,并可见脱落的肺上皮细胞。高氧损伤组与正常对照组比较逐渐表现出肺泡数目减少,直径变大,肺泡面积减小,肺泡间隔厚度减小,差异具有统计学意义(P<0.05)。
3.正常对照组第1天、第3天和第7天VEGF mRNA和蛋白的表达：正常对照组第1天、第3天和第7天VEGF mRNA相对含量分别为：1.000±0.366、0.931±0.370、0.722±0.372；正常对照组第1天、第3天和第7天VEGF蛋白相对含量分别为：1.000±0.498、0.787+0.544、0.632±0.289；正常组第1天、第3天和第7天VEGF mRNA和蛋白相对含量比较,均无显著性差异(P>0.05)。
4.正常对照组第1天、第3天和第7天Ang mRNA和蛋白的表达：正常对照组第1天、第3天和第7天Ang mRNA相对含量分别为：1.000-±1.108、0.907±1.019、0.828±0.462;正常对照组第1天、第3天和第7天Ang蛋白相对含量分别为：1.000-±0.858、0.995±0.871、0.573±0.436；正常组第1天、第3天和第7天Ang mRNA和蛋白相对含量比较,均无显著性差异(P>0.05)。
5.高氧损伤组第1天、第3天和第7天VEGF mRNA和蛋白的表达：高氧损伤组第1天、第3天和第7天VEGF mRNA相对含量分别为：0.985±0.403、0.897±0.512、0.239±0.293；高氧损伤组第1天、第3天和第7天VEGF蛋白相对含量分别为：0.960±0.619、0.730±0.342、0.358±0.128；高氧损伤组第7天新生鼠肺组织VEGF mRNA和蛋白表达水平均明显低于正常对照组第7天,存在显著差异(P<0.05)。
6.高氧损伤组第1天、第3天和第7天天Ang mRNA和蛋白的表达：高氧损伤组第1天、第3天和第7天Ang mRNA相对含量分别为：1.011±1.131、0.843±0.388、0.327±0.184；高氧损伤组第1天、第3天和第7天Ang蛋白相对含量分别为：0.755±0.414、0.711±0.539、0.204±0.068；高氧损伤组第7天新生鼠肺组织Ang mRNA和蛋白表达水平均明显低于正常对照组第7天,存在显著差异(P<0.05)。
结论
1.通过新生2-3天日龄SD大鼠持续吸入高浓度氧气(≥95%)7天可构建BPD高氧肺损伤的动物模型。
2.正常新生SD大鼠肺组织中VEGF、Ang表达呈动态变化趋势,提示VEGF、Ang在肺组织的动态表达与肺发育之间有着密切关系,进一步说明VEGF、Ang参与了肺发育过程。
3.高氧可诱导支气管肺发育不良的发生,随着高氧暴露时间的延长,高氧肺损伤时VEGF、Ang mRNA和蛋白表达均明显减少,光镜下肺组织形态逐渐呈现高氧肺损伤时肺泡发育停滞的肺部病理改变,提示高氧肺损伤时VEGF、Ang表达减少可能通过阻碍肺发育参与BPD模型鼠高氧肺损伤的发生机制,为BPD的早期干预奠定实验基础。
Bronchopulmonary dysplasia (BPD) is an important complication of premature infants, and it is a common chronic diseases of respiratory system for infants. But its certain etiological factors and pathogenesis are not clear completely, inhaling hyperoxia for a long time to lead to lung injury is a main reason, and some investigations showed that arrested lung development was the main pathological change. As well as known that immature lung development and oxygen toxicity are the key risk factors. Inhaling hyperoxia is one of the leading causes for prmature babies' bronchopulmonary dysplasia. Normal vascular development of lung includes vasculogenesis and angiogenesis. Recently, some researches considered that abnormal expression of lung development regulating factors would influence the normal morphological and vascular development of lung. Vascular endothelial growth factor(VEGF) and Angiopoietin(Ang) are two main factors which play an important role in morphological development of lung, tissue moulding and vascular remodeling. At present, it is considered that inhaling hyperoxia leading to abnormal expression of VEGF and Ang is significant in arrested lung development. Explore related pathological mechanism of VEGF and Ang in model rats with lung injury caused by hypoxia and their influence of alveoli and vascular development of lung will provide a new strategy and direction in prevention and cure of neonatal bronchopulmonary dysplasia.
Objectives
1. Make a neonatal rats BPD model by hypoxia, to observe the pathological change of lung, reveal the pathological mechanism of BPD in further to provide experimental fundation for new ways about early intervention and gene intervention therapy.
2. Explore expression characters of VEGF and Ang mRNA and protein in neonatal rats'lungs with lung injury by hyperoxia, invesigate the relationship between them and bronchopulmonary dysplasia to provide experimental fundation for research about mechanism of BPD.
Methods
1. The neonatal Sprague-Dawley rats were used as study objects after birth for2-3days, and exposured to hyperoxia(≥95%) for1,3or7days to make a model with BPD.
2. The neonatal rats were divided into contol groups and hyperoxia groups at random; lung histological changes of neonatal Sprague-Dawley rats in each group were observed and compared through light microscope. Number, diameter, area of alveoli and alveolar septum thickness were measured by image manipulation and analysis software. Quantitive analysis of histological changes were did for comparing difference of lung histomophology appearnce between groups.
3. Expression of VEGF and Ang mRNA were detected by real time fluorescent quantitation polymerase chain reaction, and the interference effect of VEGF and Ang for lung development were evaluated combined with the morphological changes of lung.
4. Protein expression of VEGF and Ang were detected by Western blotting, and the interference effect of VEGF and Ang for lung development were evaluated combined with the morphological changes of lung.
Results
1. Make a model with lung injury, BPD of neonatal rats:neonatal rats were exposed to hyperoxia(≥95%) for7days, without activity, dull reaction, their weight and length increased slowly; compared rats' weight and length of model group with control group, it was very different (P<0.05).
2. Observation of lung histological changes of rats in each group with light microscope:in low view, the lung tissue in noraml rats showed clear and alveolar structure, uniform size, no fluid exudation; the lung tissue of hyperoxia groups displayed thinner walls of alveoli, simple alveolar structure, fewer and larger alveoli, expanded and shrinked alveoli, decreased area of alveoli. In high sight, the lung tissue in normal rats showed complete walls of alveoli, no cells in alveoli; the lung tissue of model groups with hyperoxia showed few red cells, macrophages and AECⅡ in space of alveoli. Compare area, diameter of alveoli and thickness of alveolar septum of model groups with control groups, it was very different (P<0.05).
3. VEGF mRNA and protein expressions of control groups detected by real time fluorescent quantitation polymerase chain reaction and Western blotting:VEGF mRNA relative expression in control groups were1.000±0.366,0.931±0.370or0.722±0.372; VEGF protein relative expression in control groups were1.000±0.498,0.787±0.544or0.632±0.289; compared VEGF mRNA and protein expressions between them, it wasn't different (P>0.05).
4. Ang mRNA and protein expressions of control groups detected by real time fluorescent quantitation polymerase chain reaction and Western blotting:Ang mRNA relative expression in control groups were1.000±1.108,0.907±1.019or 0.828±0.462; Ang protein relative expression in control groups were1.000±0.858,0.995±0.871or0.573±0.436; compared Ang mRNA and protein expressions between them, it wasn't different (P>0.05).
5. VEGF mRNA and protein expressions of hyperoxia groups detected by real time fluorescent quantitation polymerase chain reaction and Western blotting:VEGF mRNA relative expression in control groups were0.985±0.403,0.897±0.512or0.239±0.293; VEGF protein relative expression in control groups were0.960±0.619,0.730±0.342or0.358±0.128; compared VEGF mRNA and protein expressions of the7th days between control group and hyperoxia group, it was very different (P<0.05).
6. Ang mRNA and protein expressions of hyperoxia groups detected by real time fluorescent quantitation polymerase chain reaction and Western blotting:Ang mRNA relative expression in control groups were1.011±1.131,0.843±0.388or0.327±0.184; Ang protein relative expression in control groups were0.755±0.414,0.711±0.539or0.204±0.068; compared Ang mRNA and protein expressions of the7th days between control group and hyperoxia group, it was very different (P<0.05).
Conclusions
1. Inhaling hyperoxia(≥95%) for7days persistantly could induced lung injury leading to a model of BPD.
2. VEGF and Ang mRNA and protein expression decreased with lung injury by hyperoxia, which showed that VEGF and Ang might take important part in the pathological mechanism of model rats with BPD by hyperoxia through promoting alveoli and vascular development of lung.
3. Inhaling hyperoxia persistantly could induced lung injury leading to a model of BPD, and the lung tissue of hyperoxia groups displayed thinner walls of alveoli, simple alveolar structure, fewer and larger alveoli, expanded and shrinked alveoli, decreased area of alveoli, which showed that VEGF and Ang might take an important part in lung development from lung injure of hyperoxia, and they mighty cooperate or restrict with each other..
引文
[1]Gray PH, Sarkar S, Young J, et al. Conductive hearing loss in preterm infants with bronchopulmonary dysplasia. J Paediatr Child Health,2001,37:278-82.
[2]Short EJ, Klein Nk, Lewis BA, et al. Cognitive and academic consequences of bronchopulmonary dysplasia and very low birth weight:8-year-old outcomes. Paediatrics,2003,112:e359.
[3]Jobe AJ. The new BPD:an arrest of lung development. Pediatr Res,1999,46: 641-3.
[4]金汉珍,黄德珉,官希吉主编.实用新生儿学.第三版.北京：人民卫生出版社,2003.454-461.
[5]Ferrara N, Gerber HP, LeCouter J.The biology of VEGF and its receptors.Nat Med.2003 Jun;9(6):669-76.
[6]Perl AK, Whitsett JA. Molecular mechanisms controlling lung morphogenesis. Clin Genet.1999,56(1):14-27.
[7]Warburton D, Tefft D, Mailleux A, et al. Do lung remodeling, repair, and regeneration recapitulate respiratory ontogeny? Am J Respir Crit Care Med. 2001,164:S59-62.
[8]Akeson AL, Greenberg JM, Cameron JE, et al.Temporal and spatial regulation of VEGF-A controls vascular patterning in the embryonic lung. Dev Biol.2003 Dec 15;264(2):443-55.
[9]Balasubramaniam V, Mervis CF, Maxey AM et al.Hyperoxia reduces bone marrow, circulating, and lung endothelial progenitor cells in the developing lung:implications for the pathogenesis of bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol Physiol.2007 May;292(5):L1073-84.
[10]Gale NW, Thurston G, Hackett SF, et al.Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and only the latter role is rescued by Angiopoietin-1.Dev Cell.2002 Sep;3(3):411-23.
[11]Babaei S, Teichert-Kuliszewska K, Zhang Q, e.Angiogenic actions of angiopoietin-1 require endothelium-derived nitric oxide.Am J Pathol.2003 Jun; 162(6):1927-36.
[12]Visconti RP, Richardson CD, Sato TN.Orchestration of angiogenesis and arteriovenous contribution by angiopoietins and vascular endothelial growth factor (VEGF).Proc Natl Acad Sci U S A.2002 Jun 11;99(12):8219-24.
[13]Jobe AH, Bancalarl E. Branchopulmonary dysplasia. Am J Respir Crit Care Med.2001,163:1723-1729.
[14]Remesal A, Pedraz C. Pulmonary expression of vascular endothelial growth factor (VEGF) and alveolar septation in a newborn rat model exposed to acute hypoxia and recovered under conditions of air or hyperoxia. Histol Histopathol. 2009 Mar;24(3):325-30.
[15]Coalson JJ.Pathology of new bronchopulmonary dysplasia.Semin Neonatol. 2003 Feb;8(1):73-81.
[16]Shimizukawa M, Ebina M, Narumi K,et al. Intratracheal gene transfer of decorin reduces subpleural fibroproliferation induced by bleomycin. Am J Physiol Lung Cell Mol Physiol.2003 Mar;284(3):L526-32.
[17]Abman SH.Impaired vascular endothelial growth factor signaling in the pathogenesis of neonatal pulmonary vascular disease.Adv Exp Med Biol. 2010;661:323-35.
[18]Guo Q, Jin J, VEGF, Bcl-2 and Bad regulated by angiopoietin-1 in oleic acid induced acute lung injury.Biochem Biophys Res Commun.2011 Oct 7;413(4):630-6.
[19]Mourani PM, Ivy DD, Gao D, Abman SH.Pulmonary vascular effects of inhaled nitric oxide and oxygen tension in bronchopulmonary dysplasia.Am J Respir Crit Care Med.2004 Nov 1;170(9):1006-13.
[20]Dauger S, Ferkdadji L, Saumon G, et al. Neonatal exposure to 65% oxygen durably impairs lung architecture and breathing pattern in adult mice. Chest,2003,123(2):530-538
[21]Warner BB, Stuart LA, Papes RA, et al. Functional and pathological effects of prolonged hyperoxia in neonatal mice. Am J Physiol,1998,75(1 Pt 1):1,110-117.
[22]Bhatt, D'Angio CT, Maniscalco WM.The role of vascular growth factors in hyperoxia-induced injury to the developing lung.Front Biosci.2002 Jul 1;7:d1609-23. Epub 2002 Jul 1.
[23]Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 2001,25(4):402-8.
[24]Zhang QJ, Chadderton A, Clark RL, et al. Selection of normalizer genes in conducting relative gene expression analysis of embryos. Birth Defects Res A Clin Mol Teratol,2003,67(8):533-44.
[25]Schnitzer JJ. Control and regulation of pulmonary hypoplasia associated with congenital diaphragmatic hernia. Semin Pediatr Surg JT,2004,13(1):37-43.
[26]Kumar VH, Ryan RM. Growth factors in the fetal and neonatal lung. Front Biosci JT,2004,9:464-80.
[27]Husain AN, Siddiqui NH, Stocker JT. Pathology of arrested acinar development in postsurfactant bronchopulmonary dysplasia.Hum Pathol.1998 Jul;29(7): 710-7.
[28]Akeson AL, Greenberg JM, Cameron JE, et al.Temporal and spatial regulation of VEGF-A controls vascular patterning in the embryonic lung. Dev Biol.2003 Dec 15;264(2):443-55.
[29]Lassus P, Turanlahti M, Heikkila P, et al.Pulmonary vascular endothelial growth factor and Flt-1 in fetuses, in acute and chronic lung disease, and in persistent pulmonary hypertension of the newborn. Am J Respir Crit Care Med.2001 Nov 15;164(10 Pt1):1981-7.
[30]黄培堂 等译.分子克隆实验指南.第三版.北京：科学出版社,2002.
[31]Kumar VH, Ryan RM.Growth factors in the fetal and neonatal lung.Front Biosci.2004 Jan 1;9:464-80.
[32]Bhatt AJ, Pryhuber GS, Huyck H, et al.Disrupted pulmonary vasculature and decreased vascular endothelial growth factor, Flt-1, and TIE-2 in human infants dying with bronchopulmonary dysplasia.Am J Respir Crit Care Med.2001 Nov 15;164(10 Pt 1):1971-80.
[33]Jakkula M, Le Cras TD, Gebb S, et al.Inhibition of angiogenesis decreases alveolarization in the developing rat lung.Am J Physiol Lung Cell Mol Physiol. 2000 Sep;279(3):L600-7.
[34]Le Cras TD, Markham NE, Tuder RM, et al.Treatment of newborn rats with a VEGF receptor inhibitor causes pulmonary hypertension and abnormal lung structure.Am J Physiol Lung Cell Mol Physiol.2002 Sep;283(3):L555-62.
[35]De Paepe ME, Greco D, Mao Q.Angiogenesis-related gene expression profiling in ventilated preterm human lungs.Exp Lung Res.2010 Sep;36(7):399-410.
[36]Abman SH.Bronchopulmonary dysplasia:"a vascular hypothesis".Am J Respir Crit Care Med.2001 Nov 15;164(10 Pt 1):1755-6.
[37]Gale NW, Thurston G, Hackett SF, et al.Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and only the latter role is rescued by Angiopoietin-1.Dev Cell.2002 Sep;3(3):411-23.
[38]Babaei S, Teichert-Kuliszewska K, Zhang Q, e.Angiogenic actions of angiopoietin-1 require endothelium-derived nitric oxide.Am J Pathol.2003 Jun; 162(6):1927-36.
[39]Visconti RP, Richardson CD, Sato TN.Orchestration of angiogenesis and arteriovenous contribution by angiopoietins and vascular endothelial growth factor (VEGF).Proc Natl Acad Sci U S A.2002 Jun 11;99(12):8219-24.
[40]Desai TJ, Cardoso WV.Growth factors in lung development and disease:friends or foe?Respir Res.2002;3:2.
[41]Buckley S, Warburton D.Dynamics of metalloproteinase-2 and -9, TGF-beta, and uPA activities during normoxic vs. hyperoxic alveolarization.Am J Physiol Lung Cell Mol Physiol.2002 Oct;283(4):L747-54.
[42]Supplemental Therapeutic Oxygen for Prethreshold Retinopathy Of Prematurity (STOP-ROP), a randomized, controlled trial. I:primary outcomes.Pediatrics. 2000 Feb;105(2):295-310.
[43]Jakkula M, Le Cras TD, Gebb S, et al.Inhibition of angiogenesis decreases alveolarization in the developing rat lung.Am J Physiol Lung Cell Mol Physiol. 2000 Sep;279(3):L600-7.
[2]Short EJ, Klein Nk, Lewis BA, et al. Cognitive and academic consequences of bronchopulmonary dysplasia and very low birth weight:8-year-old outcomes. Paediatrics,2003,112:e359.
[3]Jobe AJ. The new BPD:an arrest of lung development. Pediatr Res,1999,46: 641-3.
[4]金汉珍,黄德珉,官希吉主编.实用新生儿学.第三版.北京：人民卫生出版社,2003.454-461.
[5]Ferrara N, Gerber HP, LeCouter J.The biology of VEGF and its receptors.Nat Med.2003 Jun;9(6):669-76.
[6]Perl AK, Whitsett JA. Molecular mechanisms controlling lung morphogenesis. Clin Genet.1999,56(1):14-27.
[7]Warburton D, Tefft D, Mailleux A, et al. Do lung remodeling, repair, and regeneration recapitulate respiratory ontogeny? Am J Respir Crit Care Med. 2001,164:S59-62.
[8]Akeson AL, Greenberg JM, Cameron JE, et al.Temporal and spatial regulation of VEGF-A controls vascular patterning in the embryonic lung. Dev Biol.2003 Dec 15;264(2):443-55.
[9]Balasubramaniam V, Mervis CF, Maxey AM et al.Hyperoxia reduces bone marrow, circulating, and lung endothelial progenitor cells in the developing lung:implications for the pathogenesis of bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol Physiol.2007 May;292(5):L1073-84.
[10]Gale NW, Thurston G, Hackett SF, et al.Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and only the latter role is rescued by Angiopoietin-1.Dev Cell.2002 Sep;3(3):411-23.
[11]Babaei S, Teichert-Kuliszewska K, Zhang Q, e.Angiogenic actions of angiopoietin-1 require endothelium-derived nitric oxide.Am J Pathol.2003 Jun; 162(6):1927-36.
[12]Visconti RP, Richardson CD, Sato TN.Orchestration of angiogenesis and arteriovenous contribution by angiopoietins and vascular endothelial growth factor (VEGF).Proc Natl Acad Sci U S A.2002 Jun 11;99(12):8219-24.
[13]Jobe AH, Bancalarl E. Branchopulmonary dysplasia. Am J Respir Crit Care Med.2001,163:1723-1729.
[14]Remesal A, Pedraz C. Pulmonary expression of vascular endothelial growth factor (VEGF) and alveolar septation in a newborn rat model exposed to acute hypoxia and recovered under conditions of air or hyperoxia. Histol Histopathol. 2009 Mar;24(3):325-30.
[15]Coalson JJ.Pathology of new bronchopulmonary dysplasia.Semin Neonatol. 2003 Feb;8(1):73-81.
[16]Shimizukawa M, Ebina M, Narumi K,et al. Intratracheal gene transfer of decorin reduces subpleural fibroproliferation induced by bleomycin. Am J Physiol Lung Cell Mol Physiol.2003 Mar;284(3):L526-32.
[17]Abman SH.Impaired vascular endothelial growth factor signaling in the pathogenesis of neonatal pulmonary vascular disease.Adv Exp Med Biol. 2010;661:323-35.
[18]Guo Q, Jin J, VEGF, Bcl-2 and Bad regulated by angiopoietin-1 in oleic acid induced acute lung injury.Biochem Biophys Res Commun.2011 Oct 7;413(4):630-6.
[19]Mourani PM, Ivy DD, Gao D, Abman SH.Pulmonary vascular effects of inhaled nitric oxide and oxygen tension in bronchopulmonary dysplasia.Am J Respir Crit Care Med.2004 Nov 1;170(9):1006-13.
[20]Dauger S, Ferkdadji L, Saumon G, et al. Neonatal exposure to 65% oxygen durably impairs lung architecture and breathing pattern in adult mice. Chest,2003,123(2):530-538
[21]Warner BB, Stuart LA, Papes RA, et al. Functional and pathological effects of prolonged hyperoxia in neonatal mice. Am J Physiol,1998,75(1 Pt 1):1,110-117.
[22]Bhatt, D'Angio CT, Maniscalco WM.The role of vascular growth factors in hyperoxia-induced injury to the developing lung.Front Biosci.2002 Jul 1;7:d1609-23. Epub 2002 Jul 1.
[23]Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 2001,25(4):402-8.
[24]Zhang QJ, Chadderton A, Clark RL, et al. Selection of normalizer genes in conducting relative gene expression analysis of embryos. Birth Defects Res A Clin Mol Teratol,2003,67(8):533-44.
[25]Schnitzer JJ. Control and regulation of pulmonary hypoplasia associated with congenital diaphragmatic hernia. Semin Pediatr Surg JT,2004,13(1):37-43.
[26]Kumar VH, Ryan RM. Growth factors in the fetal and neonatal lung. Front Biosci JT,2004,9:464-80.
[27]Husain AN, Siddiqui NH, Stocker JT. Pathology of arrested acinar development in postsurfactant bronchopulmonary dysplasia.Hum Pathol.1998 Jul;29(7): 710-7.
[28]Akeson AL, Greenberg JM, Cameron JE, et al.Temporal and spatial regulation of VEGF-A controls vascular patterning in the embryonic lung. Dev Biol.2003 Dec 15;264(2):443-55.
[29]Lassus P, Turanlahti M, Heikkila P, et al.Pulmonary vascular endothelial growth factor and Flt-1 in fetuses, in acute and chronic lung disease, and in persistent pulmonary hypertension of the newborn. Am J Respir Crit Care Med.2001 Nov 15;164(10 Pt1):1981-7.
[30]黄培堂 等译.分子克隆实验指南.第三版.北京：科学出版社,2002.
[31]Kumar VH, Ryan RM.Growth factors in the fetal and neonatal lung.Front Biosci.2004 Jan 1;9:464-80.
[32]Bhatt AJ, Pryhuber GS, Huyck H, et al.Disrupted pulmonary vasculature and decreased vascular endothelial growth factor, Flt-1, and TIE-2 in human infants dying with bronchopulmonary dysplasia.Am J Respir Crit Care Med.2001 Nov 15;164(10 Pt 1):1971-80.
[33]Jakkula M, Le Cras TD, Gebb S, et al.Inhibition of angiogenesis decreases alveolarization in the developing rat lung.Am J Physiol Lung Cell Mol Physiol. 2000 Sep;279(3):L600-7.
[34]Le Cras TD, Markham NE, Tuder RM, et al.Treatment of newborn rats with a VEGF receptor inhibitor causes pulmonary hypertension and abnormal lung structure.Am J Physiol Lung Cell Mol Physiol.2002 Sep;283(3):L555-62.
[35]De Paepe ME, Greco D, Mao Q.Angiogenesis-related gene expression profiling in ventilated preterm human lungs.Exp Lung Res.2010 Sep;36(7):399-410.
[36]Abman SH.Bronchopulmonary dysplasia:"a vascular hypothesis".Am J Respir Crit Care Med.2001 Nov 15;164(10 Pt 1):1755-6.
[37]Gale NW, Thurston G, Hackett SF, et al.Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and only the latter role is rescued by Angiopoietin-1.Dev Cell.2002 Sep;3(3):411-23.
[38]Babaei S, Teichert-Kuliszewska K, Zhang Q, e.Angiogenic actions of angiopoietin-1 require endothelium-derived nitric oxide.Am J Pathol.2003 Jun; 162(6):1927-36.
[39]Visconti RP, Richardson CD, Sato TN.Orchestration of angiogenesis and arteriovenous contribution by angiopoietins and vascular endothelial growth factor (VEGF).Proc Natl Acad Sci U S A.2002 Jun 11;99(12):8219-24.
[40]Desai TJ, Cardoso WV.Growth factors in lung development and disease:friends or foe?Respir Res.2002;3:2.
[41]Buckley S, Warburton D.Dynamics of metalloproteinase-2 and -9, TGF-beta, and uPA activities during normoxic vs. hyperoxic alveolarization.Am J Physiol Lung Cell Mol Physiol.2002 Oct;283(4):L747-54.
[42]Supplemental Therapeutic Oxygen for Prethreshold Retinopathy Of Prematurity (STOP-ROP), a randomized, controlled trial. I:primary outcomes.Pediatrics. 2000 Feb;105(2):295-310.
[43]Jakkula M, Le Cras TD, Gebb S, et al.Inhibition of angiogenesis decreases alveolarization in the developing rat lung.Am J Physiol Lung Cell Mol Physiol. 2000 Sep;279(3):L600-7.