【作者】 张巧凤；

【导师】 富建华；

【作者基本信息】 中国医科大学 ， 新生儿急救， 2010， 硕士

【摘要】 前言随着机械通气的广泛开展,早产儿管理技术的日益提高和肺表面活性物质的普遍应用,早产儿,尤其是极低出生体重儿的存活率有了明显提高,随着而来因长时间吸入高浓度氧所导致的早产儿慢性肺疾病(chronic lung disease,CLD)的发生率亦在增加,严重影响了早产儿的生存质量,是目前导致新生儿致残主要疾病之一。尽管高氧致CLD的发生机制还不清楚,但有关其病理过程及病理变化已基本明确,即早期的肺水肿及晚期肺间质纤维化,目前的多数研究试图从拮抗肺纤维化的角度来达到目的,但效果并不理想。那么若能从CLD早期阶段—肺水肿期为突破点,探索其发生的可能机制,从而为CLD新的防治途径的设计奠定实验和理论基础。高氧肺损伤是一组织弥漫性肺泡炎和损伤后的修复重建的复杂病理、生理过程,其肺损伤早期特征性改变是肺水肿。目前认为CLD早期肺水肿的发生多与炎症因子异常表达有关,但应用其拮抗剂后其肺水肿程度无明显减轻。氧化应激反应也被认为与肺水肿有关,但应用抗氧化剂治疗和预防,疗效也不显著。水通道蛋白(Aquaporin, AQP)是新近发现与水通透性有关的细胞膜转运蛋白,目前认为与肺组织密切的有AQP1、AQP3、AQP4、AQP5。胎儿肺组织AQP1表达呈缓慢上升,而临近预产期时显著增加,生后初期随日龄呈时向性变化,提示生后初期AQP1与肺内液体快速转运密切相关。钠通道蛋白(Epithelium sodium channel, ENaC)是一种跨膜蛋白,是肺内液体转运的重要通道,由αβγ三个同源亚基组成,早产儿气道上皮ENaC表达明显低于足月儿,其低水平的表达与呼吸窘迫发生密切相关本研究采用高氧致肺损伤新生鼠模型为对象,通过测定肺组织AQP1、a-ENaC的表达基因及蛋白表达,试图阐明AQP1、a-ENaC在高氧肺损伤发生、发展中的动态表达规律。材料与方法一、动物实验(一)实验分组及动物模型的制备1、研究对象健康Wistar大鼠雌雄各40只,雌雄交配(4：1),孕22-23d自然分娩的新生鼠80只,生后12小时内(0d),依据吸氧浓度Fi02随机分组。2、动物模型建立：采用以往所建立的研究方法将新生Wistar大鼠80只依据吸氧浓度分为随机二组：实验组(Fi020.90)和对照组(Fi020.21),每组均为40只；实验组：生后即置于氧箱中,持续输入氧气,维持Fi02为0.90(用测氧仪监测),C02浓度<0.5%(用钠时会吸收C02),温度25～27℃,湿度50%70%。每天定时开箱0.5小时,添水、饲料及更换垫料,并与空气对照组交换母鼠以避免因氧中毒而致喂养能力下降。对照组：Fi02为0.21,放置与模型组同一室内空气饲养,具体方法及实验控制因素同实验组。(二)实验标本的收集、方法于实验后24h、48h、72h 5、7天分别从两组随机抽取8只,用10%水合氯醛麻醉后立即打开胸腔,分离肺组织。将左肺置于4%的多聚甲醛中固定,石蜡包埋,常规制备5um切片,苏木素-伊红染色后用于肺形态学观察。右肺组织置于无Rnase的Eppendorf管中,-80℃保存,用于免疫组化、免疫荧光、RT-PCR检测。二、实验方法及检测指标(一)肺组织形态学观察光镜下观察肺组织形态改变,观察高氧致新生鼠CLD肺的病理学改变。(二)免疫组织化学技术检测肺组织AQP1蛋白表达,免疫荧光检测肺组织α-ENaC蛋白表达于每个时间点抽取染色清晰的切片10张,每张切片于光镜下(×400)随机抽取5个视野,固定窗口面积,以胞浆中只有棕黄色颗粒为阳性细胞,其蛋白定量利用美国的Universai Imagining Porppration图像分析系统,应用Meta Morph软件测定平均积分光密度值,用来表示肺组织AQP1蛋白表达强度。每个时间点抽取染色清晰的切片10张,每张切片于光镜下(×400)随机抽取5个视野,固定窗口,于暗室中发翠绿色荧光为阳性表达,依据表达强弱来表示α-ENaC蛋白表达的强度。(三)Real-time PCR检测肺组织AQP1、α-ENaC mRNA的表达先构建目的基因(AQP1基因和a-ENaC基因)和管家基因(GAPDH)的RNA标准品,制作标准曲线,利用标准曲线对样品中的目的基因和管家基因分别进行定量。通过管家基因的校正,检测各组细胞中AQP1及a-ENaC目的基因的相对表达量。AQP1 mRNA的相对表达量=AQP1基因拷贝数／GAPDH基因拷贝数。α-ENaC mRNA的相对表达量=a-ENaC基因拷贝数／GAPDH基因拷贝数,校正结果以生理盐水对照组为1,其余组与之相比较。三统计学分析所有数据均以均数±标准差(x±s)表示,应用SPSS14.0对各组间数据进行分析,两样本方差齐性检验采用F检验,根据样本的方差齐性检验F值,两组间比较用t检验或t’检验,多组间比较采用ANOVA分析,以P<0.05表示统计学有显著意义。结果一、肺组织形态学改变对照组大鼠1-3天肺组织水肿,肺泡腔内大量渗液,间质水肿,小血管扩张充血,5-7天支气管、肺泡结构趋于正常,肺结构完整,肺泡间隔均匀一致,壁光滑,无渗出液,无出血及灶性肺水肿；高氧1-2天与对照组无明显差别；高氧暴露3天与对照组比较,见肺泡上皮细胞肿胀,肺泡腔内大量渗液,小血管扩张充血,间质水肿,炎性细胞明显增多,但肺泡结构尚完整；高氧5、7天时上述改变进一步加重,肺泡壁增厚,肺组织结构紊乱。二、肺组织免疫组织化学技术检测肺组织AQP1蛋白表达实验开始后,第24h、48h实验组与对照组肺组织AQP1蛋白表达强度无明显差异(P>0.05)。第3天AQP1蛋白表达明显低于对照组(P<0.05),高氧第5、7天实验组AQP1蛋白表达减弱更明显。(P<0.01)三、肺组织免疫荧光检测α-ENaC蛋白表达实验后,第24H、48ha-ENaC蛋白表达强度无明显差异,第3、5、7天实验组有明显减弱。四、RT-PCR技术检测肺组织AQP1mRNA、α-ENaCmRNA表达实验开始后24h、48h天,实验组和对照组肺组织AQP1mRNA、α-ENaCmRNA基因表达强度无显著差异(P>0.05)；AQP1基因表达与对照组相比于高氧后第3、5、7天减弱更加明显(P<0.05)。a-ENaC基因表达与对照组相比,第3天下降,第5、7天表达明显减弱(P<0.05)。结论1、高氧暴露后第3天AQP1蛋白及基因表达下降,第3、5、7天下降更明显,该基因蛋白表达变化与高氧3-7天出现肺水肿呈现同一时相变化。推测此时AQP1的表达下降,可能与肺水肿有关。2、高氧暴露后α-ENaCmRNA表达逐渐下降,第5、7天下降明显,也可能与肺水肿的出现有关。更多还原

【Abstract】 prefaceChronic lung disease (CLD) is a kind of syndrome caused by long time oxygen inhalation with high concentration or mechanical ventilation with high piercer. In recent years, with the conduct of assisted reproductive technology and increasingly sophisticated management techniques of premature infants, the rate of low weight infants, especially low or very low weight infants is increasing year by year, However, CLD incidence rate is also increasing, which seriously affects the quality of premature infants life. It is currently one of the major diseases leading to neonatal disability. Although the occurrence of hyperoxia-induced lung injury mechanism is unclear, the pathological changes and its pathological process has been basically clear, that is the early pulmonary edema and late pulmonary interstitial fibrosis. Most of the current study attempts to antagonize the perspective of pulmonary fibrosis to achieve their goals, but the effect is not so obvious. So if we explore the possible mechanism of its occurrence from the early stages of hyperoxia-induced lung injury to pulmonary edema, it will provide the experimental and theoretical basis for prevention and treatment of hyperoxia-induced lung injury.Hyperoxia-induced lung injury is a kind of diffuse pulmonary inflammation, injury repair and reconstruction of complex pathologic and physiological process. The early typical change of lung injury is pulmonary edema. At present, the early pulmonary edema of hyperoxia-induced lung injury is related to abnormal expression of inflammatory factors. After using its antagonist, the symptom of pulmonary edema is not significantly reduced. Oxidative stress reaction is also considered associated with pulmonary edema, but it is also not effective by application of antioxidants in the treatment and prevention.What is the the mechanism of early pulmonary edema by hyperoxia-induced lung injury?Aquaporins (AQP) are recently discovered cell membrane transporters related to water permeability.Currently AQP1, AQP3 AQP4, AQP5 are closed with the lung tissue. AQP1 expression in fetal lung tissues slowly rises, but increases significantly near the birth. In the initial period afer birth it changes timely day by day, which prompts that AQP1 is closely related with the rapid transportation of pulmonary fluid.ENaC is a trans-membrane protein and an important transport channel in pulmonary tissue, which is composed by three homologous subunitαβγThe expression of the airway epithelial ENaC in premature infants is much lower than that in normal infants. The low level expression is closely related with respiratory distress.The study is on lung injury of premature rat models exposed to hyperoxia by detection of the expression of AQP 1,α-ENaC gene and protein in lung tissue. The aim of this research is to clarify the dynamic AQP1, a-ENaC expression in hyperoxia-induced lung injury.Materials and Methods1. Animal experiment(1) Subgroup and animal model①Object of study:premature Wistar rats②he establishment of animal models:use of previously established research methodsEighty premature Wistar rats were randomly divided into two groups based on oxygen concentration level:experimental group (FiO20.90) and the control group (FiO20.21).Each group contained 40 rats.In the experimental group,the premature rats were placed in Plexiglas chamber where oxygen was continuously delivered to achieve a constant level of 90% oxygen concentration.CO2 was absorbed by sodalime to keep CO2 level below 0.5%.Temperature and humidity were maintained at 25℃-27℃and 50%-70% respectively.Chamber was opened for 0.5h daily to change water, add food and clean dirty cages.Nursing mothers were rotated between oxygen exposed and room air litters everyday to avoid oxygen toxicity.The control group was placed in air conditions (21% oxygen concentration).Methods and control factors were same with the experimental group.(2) Example collection and methodsRats from each group were killed on 24h、48h、72h、5d、7d. Thoracic cavity was opened after anesthesia with 10% chloral hydrate.Lungs were removed, and the left lungs were placed in 4% paraformaldehyde for hematoxylin-eosin stain an immunohistochemistry.2.Experimental methods and test indicators(1) histomorphologyObserve lung histomorphology using light microscope to invest the pathology of hyperoxia-induced lung injury.(2) The immunohistochemistry of lung (AQP1) and the immunofluorescence technology of lung (a-ENaC)10 slices were selected randomly at different time points and five fields of every slice was selected randomly using light microscope.Windows were fixed if buffy grains were founded in endochylema and the cell was positive. American universal imaging porppration system was used to analyse the quantitative determination while Meta Morph software was used to accumulate the mean density value.(3) AQP1mRNA in lung tissue detecting using RT-PCR technologyThe RNA of destinated gene(AQP1 gene and a-ENaC gene)and house-keeping gene(GAPDH) was constructed first while standard curve was also made. Destinated gene and house-keeping gene were quantitated by standard curve respectively. The relative expression of AQP1 gene and a-ENaC gene was detected by the rectification of house-keeping gene in cells of all groups. The expression of AQP1 mRNA=AQP1 gene copy/GAPDH gene copy. The expression of a-ENaC mRNA=a-ENaC gene copy /GAPDH gene copy.The results were corrected by the control group with physiologic saline. 3.atistical analysisSPSS 14.0 was used to perform statistical analysis,with all data expressed as (x±S), according to the test of homogeneity.F-test or f-test was used in two groups and spearman was to analyse the correlation.ANOVA was used to analyse multigroups and it had significance when P<0.05.Results1.The histmorphology findings of lung tissue24 hours of the experiment,it was observed in both room air and hyperoxia group that the alveolar was thick and the alveolar structure was irregular.On day2,3, the alveolar septum was thinner and the alveolar of the air group was more regular meanwhile in hyperoxia group there was inflammatory response,more RBC in room..In hyperoxia group on day 5-7,it was observed that there was inflammatory response,more interstitial cells, lung septum degarded and lung edema meanwhile in air group alveolar septum much thinner and the alveolar structure was graduatelly irregular and there was inflammatory response and so on.2.The expression of AQP1 protein in the lungsIn the experiment, on day land 2,there was no significant deviation in the expression strength of AQP1 protein between two groups(P>0.05).On day 3,the expression was9.01±8.95 in experiment group and 13.97±6.78 in air group.on day 5..5.42±2.60 in experiment group,14.08±6.84 in air group, On day 7,6.69±8.53 in experiment group and13.93±8.28 in air group (there was light recovery.There was significant deviation between two groups (P<0.05)3.a-ENaC in lung tissue detecting using Immunofluorescence technologyIn the experiment, on day land 2,there was no significant deviation in the expression strength of a-ENaC protein between two groups.On day 3,5and 7,the expression strength of a-ENaC protein was gradually decreased in experiment group4. AQP1mRNA、α-ENaCmRNA in lung tissue detecting using RT-PCR technologyThe expression of the lung tissue AQP1mRNA was 8.20±5.14,11.76±8.66, 9.01±8.95,5.42±2.60,6.69±8.53 in the experiment group and 8.63±4.14 14.09±13.54 13.97±6.78 14.08±6.84 13.93±8.28 in the air group on 24h、48h、72h、5d、7dThe expression of the lung tissue a-ENaC mRNA was 0.89±0.16,0.83±0.14, 0.69±0.17,0.63±0.17,0.66±0.15 in the experiment group and 0.91±0.30, 0.88±0.15,0.89±0.21,0.90±0.21,0.88±0.20 in the air group on 24h、48h、72h、5d、7dConclusion1.The expression of AQP1 and mRNA in lung tissue of rats exposed to hyperoxia was lower on day3 and much lower on day5. On day7, it slightly recovered.There was significant deviation on day 3,5,7.2.The expression ofα-ENaC mRNA in lung tissue of rats exposed to hyperoxia was gradually decreased; there was significant deviation on day5,7 and it had statistically significant.更多还原