Hoxa5、Hoxb5基因在高氧致CLD新生大鼠肺中表达及其与肺泡上皮细胞损伤修复障碍的关系
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摘要
前言
早产儿慢性肺疾病(chroniclungdisease,CLD)是早产儿长时间吸入高浓度氧或机械通气治疗或感染后最常见的和最严重的并发症。早产儿CLD作为早产儿肺透明膜病治疗的并发症在1967年首次被Northway描述,也称为支气管肺发育不良(bronchopulmonary dysplasia,BPD)。氧疗法是患有心肺疾病的早产儿最常应用的一种治疗手段。这一措施虽可改变患儿的低血氧状态、挽救患儿的生命,但长期吸入高浓度的氧气,可引起不同程度的肺损伤,严重者可发展为CLD。随着机械通气的广泛开展、早产儿管理技术的日益提高及肺表面活性物质的普遍应用,使早产儿,特别是超低出生体重儿(extremely low birth weight, ELBW)(出生体重<1000g存活率有了明显改善,但随之而来的是早产儿慢性肺疾病发生率的提高,国外已达30%-40%,国内也有上升趋势。由于尚缺乏有效的监控和防治手段,其中10%-15%CLD患儿因严重肺功能障碍而死于呼吸衰竭,存活者也需长期依赖氧气或机械通气治疗。
初期肺泡上皮细胞(alveolar epithelial cell,AEC)损伤和晚期肺纤维化是CLD主要病理特征。以往的研究侧重阐明CLD的AEC损伤(坏死或调亡)及其损伤后纤维组织替代AEC的病理过程及其可能机制,故目前多试图通过减轻AEC损伤程度或减少肺组织胶原沉积而达到有效防止CLD的目的,但效果均不理想,至今在CLD防治措施方面的研究进展缓慢。但恰是这种正常肺上皮重塑障碍后的不可逆肺纤维化,严重影响了患儿的呼吸功能。那么损伤后的肺泡上皮为何不能完全修复而被纤维组织替代呢?目前尚无答案。因此,我们设想如以研究高氧致CLD中AEC的修复及其调控机制为切入点,可能是解决肺上皮重塑障碍的重要突破口。
AEC包括Ⅰ型肺泡上皮细胞(typeⅠalveolar epithelial cell, AEC-Ⅰ)和Ⅱ型肺泡上皮细胞(typeⅡalveolar epithelial cell, AEC-Ⅱ),已证实AEC-Ⅰ为终末已分化细胞,无再生、增殖及分化能力；目前研究证实AEC-Ⅱ是AEC-Ⅰ的干细胞或祖细胞,如体外培养的AEC-Ⅱ,可很快失去其特有的标志SPC(surfactant proteinC),而表达AEC-Ⅰ的特有标志AQP5 (aquaporin5);用3H-TDR标记动物体内的AEC-Ⅱ,2天后在AEC-Ⅱ临近发现标记的AEC-Ⅰ等。由此可见,若肺上皮损伤后,AEC-Ⅰ无再生、修复能力,完全依赖AEC-Ⅱ的增殖、分化方能完成正常的肺上皮的重塑。
近年来,由于Hox基因(homeobox genes, Hox genes)在进化过程中的极大保守性和其在动物胚胎发育分化中的作用,使之成为当今世界生命科学最热门的研究领域之一。而有关Hox基因对正常肺组织发育及细胞分化调控作用的研究尚处起步阶段。多数文献阐明不同簇的Hox基因在胎肺发育的特定阶段、特定部位表达,对肺细胞系分化、成熟期重要作用,如HoxA5和HoxB5与支气管分枝形成,HoxB7、HoxB8和HoxB5与肺芽发育密切相关。有个别报道HoxB5异常表达与先天性肺隔离症、HoxA5过渡表达与原发性肺动脉高压、肺气肿发生有关。Hox基因对生后肺发育的调控机制目前尚不清楚,生后肺组织中仍存在Hox基因,其中表达最强的是HoxA5,,其次是HoxB5,且这两种基因主要定位于肺泡上皮细胞,而AEC-Ⅰ又为终末已分化细胞,故我们推测,高氧CLD时,能否因AEC-Ⅱ的HoxA5、HoxB5基因的表达降低而导致其向AEC-Ⅰ的分化障碍。
我们假设：HoxA5和HoxB5基因可能是AEC-Ⅱ向AEC-Ⅰ分化的主控基因之一,其表达异常导致了损伤AEC-Ⅰ的修复障碍,最终发生肺上皮再生受抑,纤维组织增生替代的病理结局。如该假设成立,不仅可以阐明在正常早产鼠肺发育及CLD发生、发展中HoxA5、HoxB5时空表达模式,更重要的是发现Hox同源盒基因对AEC-Ⅱ分化的调控作用,从而为进一步探索有效防治早产儿CLD的新途径奠定理论和实验基础。
实验材料与方法
一、动物模型
本实验是在中国医科大学盛京医院实验动物中心完成。足月新生Wistar大鼠(连同母鼠)被随机分为2组：A组即试验组、单纯高氧暴露；B组即对照组、正常空气吸入；将A组在生后24 h内置于玻璃氧舱中,持续输入氧气,维持在FiO20.85,每日两次监测氧浓度(美国OM-25ME型测氧仪监测),用钙石灰吸收CO2,使其浓度小于0.5%(美国Dapex气体分析仪),温度20℃-25℃,湿度50%-70%,每天开箱0.5h,称重,添加水及饲料,更换垫料,与空气组互换母鼠以避免母鼠因氧中毒而致护理能力降低；空气对照组置于空气中(Fi020.21),饲养条件与高氧组相同。
二、标本采集和处理
分别于实验开始后的第1、3、7、14、21天分别从每组中随机抽取8只新生鼠,腹腔注射5%水合氯醛6ml/kg麻醉后处死。立即打开胸腔,无菌条件下分离肺组织,取右肺各叶置于无Rnase的Eppendorf管中于-80℃冰箱中保存。取左肺置于0.1%DEPC的4%多聚甲醛(0.1MPBS, pH7.0-7.6)固定,常规脱水、石蜡包埋。
三、实验方法
1、动态观察各组新生大鼠的一般状态、监测体重。
2、病理形态学研究
(1)光镜观察：切片进行常规HE染色,光镜下动态观察肺组织的病理改变。
(2)肺泡面积/肺间隔面积(A/S)(美国Universal Ima-grog Porporation图像分析系统)。
(3)放射状肺泡计数(radical alveolar counts, RAC)。
(4)透射电镜观察AEC-Ⅱ超微结构改变。
3、采用免疫组化(Immonohistochemistry, IH)、RT-PCR检测肺组织AQP5、SPC的表达
4、两组肺组织HoxA5、HoxB5蛋白含量和基因表达的动态变化
(1)免疫组化方法检测肺组织HoxA5、HoxB5在肺组织中表达的强度和部位。
(2) RT-PCR技术检测肺组织HoxA5、HoxB5mRNA的表达。
(3)原位杂交(Hybridization in situ, ISH)检测表达HoxA5、HoxB5mRNA强度和部位。
(4)Western-blot (WB)方法检测肺组织HoxA5、HoxB5蛋白定量。
四、统计学分析
应用SPSS13.0统计软件进行统计学处理,数据以均值±标准差(x±s)表示,单因素多水平比较采用One Way ANOVA。两样本均数间比较采用Independent t test。相关分析采用Spearman分析。结果以P<0.05有意义。
结果
一、实验组和对照组新生大鼠一般状态比较
实验组新生鼠1d肤色红润,脱离氧气无呼吸困难,3d离氧后出现呼吸急促,一般在7d后出现少动、呼吸急促明显、皮肤苍白以及离氧后不同程度的发绀,随高氧暴露时间延长而逐渐加重,在14d后出现对氧的依赖。而对照组组则无以上异常表现。
实验组在高氧暴露7d开始,新生大鼠体重与对照组相比下降(P<0.05),随着高氧暴露时间的延长,体重差异逐渐明显(P<0.01)。
二、两组新生大鼠肺组织病理形态学变化的比较
1、肺组织的病理改变：
对照组1d的肺组织出现小而浅的原始肺泡,形态不规则,肺泡间隔较厚；3d时终末气腔大小降低,数量渐增多,肺泡间隔渐薄；7d-21d肺泡化进一步完善,到21d肺泡间隔变薄,肺泡次级隔明显增多,肺泡数量增多,形态较规则；实验组在高氧暴露1d后与对照组无明显差别。3d可见小血管扩张、充血,肺泡腔内或间隔少量出血,以中性粒细胞为主的渗出、间质内细胞增多。7d时炎症反应进一步加重,肺泡间隔水肿变宽,出现终末气腔扩张、肺泡融合、肺泡分隔减少等。14d-21d间隔增宽,小灶或多灶状实变,肺组织正常结构破坏,且终末气腔扩张更为明显、小肺泡数量更少,肺泡次级隔明显减少。
2、A/S和RAC动态变化：
生后1d和3d,两组A/S和RAC均无差异(p>0.05)。7d,14d,实验组的A/S、RAC明显低于对照组(p<0.01),A/S高于对照组(p<0.01),21d时A/S升至最高和RAC降至最低(p<0.001)。
3、肺泡Ⅱ型上皮细胞超微结构改变
正常AEC-Ⅱ的表面有大量微绒毛,细胞结构完整,胞核呈椭圆形,核膜完整,染色质分布均匀；胞浆内含有线粒体和大量板层小体。吸高氧后1d,即AEC-Ⅱ的少数线粒体肿胀；3d时LB开始有排空现象；7d时细胞胞膜上微绒毛减少,胞核尚完整,染色质以异染色质为主,线粒体肿胀和板层小体空泡化；14d时,游离端大量微绒毛突出细胞表面并少量脱落,线粒体嵴断裂,部分溶解,半层小体基本排空；21d细胞核固缩、溶解,线粒体、板层小体、微绒毛结构均消失。
三、高氧致慢性肺疾病新生鼠肺泡上皮细胞SPC和AQP5表达及动态变化
1、高氧后新生大鼠肺组织SPC、AQP5蛋白表达的动态变化
免疫组化结果示：SPC为Ⅱ型肺泡上皮细胞标志蛋白,主要在胞浆中表达。实验组3d明显低于对照组(P<0.05),1d、7d无明显差异,14d、21d明显高于对照组(P<0.05)；AQP5为Ⅰ型肺泡上皮细胞的标志蛋白,胞浆、胞膜可见棕黄色染色。其表达1d、3d两组无明显差异,7d、14、21d实验组均低于对照组,差异显著(P<0.05)。
2、高氧后新生大鼠肺组织SPC、AQP5mRNA表达动态变化
SPCmRNA的表达不呈直线改变,实验组与对照组比较,1d两组间无明显差异,3d时实验组明显减少有显著差异,7d、14d、21d时其表达有明显增多,且相对量多于对照组,两组间差异不显著(P>0.05)。AQP5mRNA表达量实验组逐渐减少,14d、21d变化减缓；对照组逐渐增加且趋势平缓。两组间除1d外其他各组均存在显著差异(P<0.05)。
四、高氧致CLD新生大鼠肺组织HoxA5和HoxB5基因表达动态变化的实验研究
1、高氧后新生大鼠肺组织HoxA5、HoxB5蛋白表达的动态变化
免疫组化结果显示HoxA5在新生鼠肺组织中主要表达肺泡上皮细胞、肺间质细胞、血管内皮细胞,少量表达于支气管粘膜细胞。随着肺泡化的发生,其在肺泡上皮细胞表达增多趋势更明显。对照组1d、3d表达量均较多且无明显差别(P>0.05),7d后平稳表达并且维持在较高水平。实验组,1d、3d与对照组表达量无明显差异(P>0.05),7d开始表达量逐渐下降,14d表达最低,21d表达仍低于对照组(P<0.05)。HoxB5在新生鼠肺组织中主要表达于肺泡上皮细胞、支气管粘膜细胞、血管粘膜及粘膜下,肺间质亦有少量表达。随着肺泡化的发生,其在肺泡间隔及肺泡嵴上的表达也越明显。对照组1d、3d表达量均较多且无明显差别,7d表达达高峰,14d、21d平稳表达并且维持在较高水平。实验组,1d、3d与对照组表达量无明显差异(p>0.05),7d开始表达量逐渐下降,7d、14d、21d与对照组相比差异显著(P<0.05)。
Western结果与免疫组化结果基本一致,对照组HoxA5蛋白1d、3d表达较多,7d、14d、21d表达增多。实验组HoxA5蛋白1d、3d表达较多,7d、14d、21d表达减少且与对照组存在显著差异(P<0.05)。HoxB5蛋白1d、3d表达较多,7d、14d、21d表达增多。实验组HoxB5蛋白1d、3d表达较多,7d、14d、21d表达逐渐减少且与对照组存在显著差异(P<0.05),21d时其蛋白表达基本为阴性。
2、高氧后新生大鼠肺组织HoxA5、HoxB5mRNA表达的动态变化
RT-PCR结果显示：对照组HoxA5mRNA1d、3d表达较多,7d、14d、21d表达增多。实验组HoxA5mRNAld、3d表达较多,7d、14d、21d表达逐渐减少且与对照组存在显著差异(P<0.05)。HoxB5mRNA在新生鼠肺组织中表达,实验组与对照组之间1d、3d无明显差异(P>0.05),表达较多,而7d、14d、21d差异显著(P<0.05)。实验组7d之后HoxB5mRNA表达逐渐减少,至21d时表达已极其微弱,对照组表达的量逐渐增多,14d后表达平缓,7d、14d、21d两组间存在显著差异(P<0.05)。
原位杂交结果：HoxA5 mRNA在新生鼠肺组织中主要表达于肺泡上皮细胞、肺间质细胞、血管上皮细胞,支气管粘膜上皮细胞也有少量表达。在细胞核和／胞浆中均有表达,1d、3d时肺泡间隔细胞和肺泡上皮细胞表达均较多,随着肺泡化的发生,其在肺泡上皮细胞表达增多趋势更明显,实验组3d细胞核表达量较多。对照组1d、3d表达量均较多且无明显差别(P>0.05),7d后平稳表达并且维持在较高水平。实验组,1d、3d与对照组表达量无明显差异(P>0.05),7d开始表达量逐渐下降,21d表达很少,7d、14d、21d两组差异显著(P<0.05)。HoxB5 mRNA在新生鼠肺组织中主要表达于肺泡上皮细胞、支气管粘膜细胞、血管粘膜及粘膜下,肺间质亦有少量表达。随着肺泡化的发生,其在肺泡间隔、肺泡连接处及肺泡嵴上的表达也越明显。对照组1d、3d表达量均较多且无明显差别(P>0.05),7d表达达高峰,14d、21d平稳表达并且维持在较高水平。实验组,1d与对照组表达量无明显差异(P>0.05),3d时表达已开始减少,7d、14d、21d表达水平均较低,与对照组差异明显(P<0.05)。
结论
1、新生大鼠持续高浓度氧气暴露,可导致新生大鼠肺组织出现肺泡发育障碍和肺纤维组织增生等病理形态学改变,符合早产儿CLD的发生发展特点和解剖学改变。
2、高氧肺损伤肺泡发育停滞或者肺泡损伤后修复障碍在新生鼠CLD早期即有明显表现,随着新生鼠高氧暴露时间的延长这一特点更加明显。
3、新生大鼠肺组织SPC、AQP5主要表达于肺泡上皮细胞。AQP5在支气管粘膜和小血管粘膜下层亦有表达。高氧7d后SPC表达量增多但是表达较紊乱,肺泡间隔细胞表达较多。
4、根据肺泡上皮细胞特异性标志物的变化,高氧致肺损伤,肺泡上皮细胞改变可能为：AEC-Ⅱ早期减少,随后增加并维持在较高水平；AEC-Ⅰ进行性减少。
5、既然AEC-Ⅱ是AEC-Ⅰ的干细胞,推测高氧致新生鼠CLD肺泡损伤时可能存在AEC-Ⅱ向AEC-Ⅰ的分化障碍。
6、HoxA5和HoxB5基因在正常新生鼠肺组织中表达,在肺泡形成期,表达量呈上升趋势。两个基因均在肺泡上皮中表达,在肺泡嵴、肺泡拐角、肺泡连接处表达更明显。
7、高氧致新生鼠肺损伤导致HoxA5、HoxB5基因表达异常,且随着暴露时间的延长,其表达量渐低。
8、高氧致肺损伤早期肺泡上皮损伤,肺泡修复障碍,HoxA5、HoxB5基因可能调控AEC-Ⅱ转化为AEC-Ⅰ。
Introduction
Chronic lung disease (CLD) of prematurity is the most frequent and severe complication that is caused by inhaling concentration oxygen, mechanical ventilation therapy or lung infection. Bronchopulmonary dysplasia (BPD) is one of chronic lung diseases first described in 1967 as a complication of therapy for premature infants with hyaline membrane disease. At the same time treatment with high concentration of oxygen was thought to be a major contributor to its development. Since administration of supplemental oxygen as one of supportive therapies elevates oxygen concentrations, that may cause pulmonary oxygen toxicity, and even develop to CLD. Improvements in respiratory care and management had allowed infants of more than 30 wk of gestation to survive their respiratory distress syndrome, even before the widespread useof exogenous surfactant therapy. This had led to an increased survival of very immature infants (BW<1000g), who were at most risk of developing bronchopulmonary dysplasia.(reaching 30%-40% abroad, upgrading tendency domestic). In total about 10%-15% CLD infants died with respiratory failure due to lung functional disturbance, because of deficiency of effect monitoring and treating means. Consequently the survival have to rely on oxygen or mechanical ventilation for a long term.
Pulmonary fibrosis has happened followed with alveolar epithelial cell (AEC) damage which is the main pathology characteristic. Formerly, lots of studies tried to illuminate the mechanism of the alveolar epithelial cell impairment (necrosis or apoptosis) and the pathological process of fibrous tissue substituting AEC. In order to prevent CLD many experiments have been done to relieve the degree of AEC injury and the amount of collagen deposition. Unfortunately there has no effective measures.So far the development of the research about CLD treatment is still on the slow pace. It is just the inreversible pulmonary fibrosis after the normal alveolar epithelial cell defeated in remodeling that impacts the infants'respiratory function. Why the damaged AEC couldn't be repaired but substituted by fibrous tissue? There is no answer for it. So we assume that studying the reparation of AEC and the mechanism of its regulation might be the break to resolve the problem about the normal alveolar epithelial cell defeated in remodeling.
The alveolar epithelial cells consist of typeⅠ(AEC-Ⅰ) and typeⅡ(AEC-Ⅱ)cells. It has been confirmed that typeⅠalveolar(epithelial)cells (AEC-Ⅰ) are the noble cells which can't be regenerated, proliferated and differentiated. Meanwhile typeⅡalveolar (epithelial) cells (AEC-Ⅱ) are the main stem cells in the lung. If AEC-Ⅱare cultured in vitro they will lose their specific marker SPC while expressing aquaporin5 (AQP5) which is the specific marker of AEC-Ⅰ. After 3H-TDR tagged animal endosomatic AEC-Ⅱfor 2 days, the labelled AEC-I would be found near the AEC-Ⅱ. It can be seen that if the alveolar epithelial cells are damaged it would depend on AEC-Ⅱto proliferate and differentiate to accomplish the epithelial remodeling as AEC-I hasn't the ability of regeneration and reparation.
For the past few years the function of homeobox genes (Hox genes) in embryonic development and differentiation has been one of the hot topics all around the world because of Hox genes'conservatism in evolutionary process. The study about hox genes in normal lung development and its functionary in pneumocyte differentiation is in early stage. Different Hox genes are expressed in different regions during embryonic lung development which are important to lung cell lines differentiation and maturation. For example, there is an intimate correlation between hoxa5 and hoxb5 and branching morphogenesis, and so is hoxb7, hoxb8 and hoxb5 and lung bud development. Several reports have described that the normal expression of hoxb5 is concerned with congenital pulmonary sequestration and the excessive expression of hoxb5 is concerned with primary pulmonary hypertension and emphysema. The mechanism of the regulation of hox genes expression in the post-lung has not been clear. Hox genes are also expressed in the neonatal lung,. The expression of hoxa5 is the most intensive followed by hoxb5. The two genes are mainly located in alveolar epithelial cells, and AEC-Ⅰis the ultimate differentiated cell.So we assume that the decrease of expression of hoxa5 and hoxb5 might be the cause of dysdifferentiation of AEC-Ⅱto AEC-Ⅰin the hyperoxia induced CLD.
We have a hypothesis:hoxa5 and hoxb5 may be two of the chief genes that control AEC-Ⅱdifferentiated to AEC-Ⅰ.The abnormal expression of them leads to failure of the injured AEC-Ⅰreparation, and results in the ultimate pathology of restrained reepithlialization at last and fibrous tissue substituting the epithelium. If the hypothesis was correct we could illuminate the time-space model of hoxa5 and hoxb5 genes'expression in the normal and CLD rats'lungs but also we could find out that hox genes might play a role in the regulation of the AEC-Ⅱdifferentiation. Thus it will provide experimental and theoretical basis to accomplish the pathogenesis and preventive and therapeutic methods of CLD in premature.
Materials and Methods
ⅠAnimal model
The present study was performed in accordance with the guidelines provided by the Experimental Animal Laboratory of Shengjing Hospital China Medical University. Within 24 hours after birth, pups were randomly redistributed to the newly delivered mothers. Term neonatal Wistar rats were divided randomly into two groups.A gruop is an experimental group,which was exposed to oxygen. B group is AIR group that served as controls and were kept in air and not subject to hyperoxia. The pups of hyperoxia exposure groups were contained in glass chambers in which the oxygen was infused continuously to achieve 85% oxygen concentration which was monitored with an oxygen monitor(OM-25ME, USA) twice daily. CO2 was removed by soda lime absorption to keep CO2 levels below 0.5%(Dapex Gas Monitor, USA). Temperature and Humidity was maintained at 20℃-25℃and 50%-70%respectively. We opened the chambers for 0.5h to exchange nursing mothers between air and O2-exposed groups every 24 hours to prevent maternal O2 toxicity and eliminate maternal effects between groups. Meanwhile change water, add food, clean dirty cages and record body weight. All animals were raised in the same room and all other conditions were the same.
ⅡTissue preparation
On postnatal 1d,3d 7d,14d,21d. Each group of hyperoxia as well as air rats were anesthetized by intraperitoneal injection of 5% chloral hydrate (6ml/kg), and put to death. The thorax was opened and the lungs were resected. The right lung tissues were put in the Rnase-free Ependorf tubes, and immediately frozen in liquid N2 for mRNA, Enzyme linked immunoadsorbent assay(ELISA), and so on. The left lung tissues were fixed in 4%formaldehydum polymerisatum which contained 0.1% DEPC(0.1MPBS, pH7.0-7.6), then were dehydrated, embedded in wax within 24 hours.
ⅢExperiment methods
1.The appearances and weight were monitored daily in each experiment groups.
2.The changes of the lung pathology
(1)For lung pathology assessment, lung sections from the left lobes were stained with HE (hematoxylin and eosin).
(2)Ratio of alveolar and septa area(A/S).
(3)Radical alveolar counts(RAC).
(4)Electronic microscopy:the observation of the ultrastructures of AECII.
3.Using Immonohistochemistry and Reverse transcription polymerase chain reaction(RT-PCR) to detect protein and mRNA of AQP5 and SPC expression in the lung.
4.The measurement of protein and gene level of HoxA5 and HoxB5 dynamic expression in the lung tissue
(1)Immonohistochemistry:the detection of intensity and position of the protein expression in lung tissue about HoxA5 and HoxB5
(2)RT-PCR:the detection at mRNA level of HoxA5 and HoxB5 in the lung tissue.
(3)Hybridization in situ:the detection of intensity and position of the mRNA expression in lung tissue about HoxA5 and HoxB5
(4)Western-blot:the detiction at the protein level of HoxA5 and HoxB5 in the lung tissue of neonates rats.
IV Statistical analysis
SPSS versionl3.0 was used to perform statistical analysis with all data expressed by (mean±SD). Statistically significant differences in the mean values among multiple groups were analyzed using One Way ANOVA analysis.The mean value of two groups were analyzed with Independent-Samples T Test. Correlation between two variables was analyzed with Spearman analysis. Statistically significant differences set P<0.05.
Results
ⅠGeneral status of rats in experimental groups and control groups
The hyperoxia rats were didn't present dyspnea after one day of oxygen exposure. Tachypnea was observed on the 3rd day after they were detached from oxygen supplementation. After the 7th day of oxygen exposure they began to appear fatigue, pale, and to present tachypnea and cyanosis for different degrees. After 14 days some even had to rely on high oxygen and become worse with the time gone. The control groups didn't have the appearances above.
At the beginning there was no difference in average birth weight between the two groups. From 7 days of oxygen exposure, weights of the experimental groups showed a significant decrease compared with control groups at the sams time period(P<0.05).
ⅡComparision of lung pathomorphism of the two neonatal rats' groups
1. The pathological changes of the lung tissue:In the control groups,on the 1st day of the experiment, it was observed that the alveolar structure was irregular, terminal air space size was rather small, and the alveolar septum was thick. On the 3rd day, the alveolar structure of each group was more regular, the size of alveolus was equal, and alveolar septum was thinner, but in the experimental group, there were a few inflammatory cells exuded out, and blooding interstitial cells increased. On the 7th day the alveolar size was equal in room air rats, while the terminal air space size of the oxygen-exposed rats became large, and there was inflammatory response and more interstitial cells. On 14th day and 21st day, air groups continued to develop alveolization, the alveolar became more regular, the size of alveolus was equal and the alveolar septum was thinner; but in O2 groups, the terminal air space size grew significantly large, secondary septum decreased, the quantity of alveolar reduced with alveolus fusion, interstitial cells increased, and alveolar septa was thicker. On the whole the severity of pathological changes was parallel to the duration of exposure.
2. The dynamic changes of ratio of alveolar and septa area(A/S) and Radical alveolar counts(RAC):No significant differences about A/S and RAC could be seen between experimental groups and control groups on day 1-3(p>0.05). But there was a significant difference between the experimental groups and the control groups on the 7th and 14th day(p<0.01). On the 21st day, A/S increased to the highest level and RAC decreased to the lowest level.
3. Ultrastructure changes of AECⅡ:There were large normal microvilli (Mv) on the surface of AECⅡ, the structure of the cell was in integrity, the nucleus was ellipse, nuclear membrane was complete, and chromoplasm was distributed evenly; There were mitochondrias(Mi) and large number lamellar bodies(LB) in the intracytoplasm. After exposure hyperoxia for one day, some mitochondrias were swelling. LB began to be emptied on the 3rd day; and on the 7th day the number of Mv was decreased,the main chromoplasm was heterochromatin, Mi swelled and LB was vacuolization, but the nucleus was complete. On the 14th day large number of microvilli outpocketed from the membrane and several of them ablated. Mitochondrial cristae broke and some of them dissolved. On the 21st day there was karyopyknosis and cell nucleus dissolved,and d Mi, LB and Mv disappeared.
Ⅲ.The dynamic changes of SPC and AQP5 expression in the lung tissue of hyperoxia induced CLD
1. The dynamic changes of SPC and AQP5 protein expression in the neonate rat lung after being exposed in hyperoxia
Immuonhistochemical studies shows SPC is an identification marker of AECⅡ, which is expressed in kytoplasm of the typeⅡalveolar epithelial cells in the lung. Compared with the control group, SPC expression in the experimental group decreased significantly on the 3rd day (P<0.05) while it increased obviously on the 14th and 21st day (P<0.05),but there was no difference on the 1st and 7th day. AQP5 is an identification marker of AECI, which is expressed in kytoplasm and cell membrane of the typeⅠalveolar epithelial cells in the lung. On the 1st and 3rd day, there was no difference of its expression, but on the 7th,14th and 21st day its expression in the experimental group decreased significantly (P<0.05)
2. The dynamic changes of SPC and AQP5 mRNA expression in the neonate rat lung after being exposed in hyperoxia
The expression tendency of SPC mRNA was not in a straight line.Compared with the control group, SPC mRNA expression in the experimental group decreased significantly on the 3rd day (P<0.05),while it increased obviously on the 7th,14th and 21st day (P<0.05) and its relative amount is more than that in the control group, but there was no difference on the 1st day. AQP5 mRNA expression in the experimental group decreased gradually, and its change slowed down on the 14th and 21st day. The level of AQP5mRNA in the control group increased gradually and its tendency was gentle. There was significant difference between the two groups except the 1st day.
IV Empirical study of the dynamic changes of HoxA5 and HoxB5 genes expression in the lung tissue of hyperoxia induced CLD
1. The dynamic changes of HoxA5 and HoxB5 protein expression in the neonate rat lung after being exposed in hyperoxia
Immuonhistochemical results showed that HoxA5 protein was mainly expressed in alveolar epithelial cells, mesenchymal cells and vascular endothelial cells, and small amounts of them expressed tunica mucosa bronchiorum cells in the lung. With the alveolization development, the tendency of HoxA5 protein expression was more obvious in alveolar epithelial cells. In the control group, the level of HoxA5 protein expression was high and there was no significant difference from the 1st to 3rd day(p> 0.05), and its expression was steady and kept on a high level after 7 days. Compared with the control group, HoxA5 protein expression in the experimental group began to decrease on the 7th day and got to the lowest level on the 14th day, and it remained on a low level on the 21st day (P<0.05),while there was no significant difference between the 1st and 3rd day(p>0.05). HoxB5 protein was mainly expressed in alveolar epithelial cells, tunica mucosa bronchiorum cells and vascular endothelial cells, and small amounts of them expressed in mesenchymal cells of the neonate rat lungs. With the alveolization development, more HoxB5 protein was obviously expressed in the alveolar septum and cristae. In the control group, the level of HoxB5 protein expression was high and there was no significant difference between the 1st and 3rd day, it reached the peak on the 7th day, and its expression was steady and kept on a high level on the 14th day and 21st day. Compared with the control group, there was no significant difference of HoxB5 protein expression between the 1st and 3rd day(p>0.05). HoxB5 protein expression decreased gradually on the 7th day, and the difference of HoxB5 protein expression was significant between the two groups on the 7th,14th and 21st day (P<0.05)
Western blotting got the similar results as those in imunohistochemistry. In the control group, the level of HoxA5 protein expression was high and there was no significant difference between the 1st and 3rd day(p>0.05), HoxA5 protein expression increased on the 7th,14th and 21st day. In the experimental group, the level of HoxA5 protein expression was high on the 1st and 3rd day, HoxA5 protein expression decreased and there was significant difference between the two groups on the 7th,14th and 21st day (P<0.05). In the control group, the level of HoxB5 protein expression was high on the 1st and 3rd day, HoxB5 protein expression increased on the 7th,14th and 21st day. In the experimental group, the level of HoxB5 protein expression was high on the 1st and 3rd day, HoxB5 protein expression decreased gradually and there was significant difference between the two groups on the 7th,14th and 21st day (P<0.05), and the protein expression of HoxB5 was almost negative on the 21st day.
2 The dynamic changes of HoxA5 and HoxB5 mRNA expression in the neonate rat lung after being exposed in hyperoxia
RT-PCR results showed:In the control group, the level of HoxA5 mRNA expression was high on the 1st and 3 rd day, and HoxA5 mRNA expression increased on the 7th,14th and 21st day. In the experimental group, the level of HoxA5 mRNA expression was high on the 1st and 3rd day, HoxA5 mRNA expression decreased gradually and there was significant difference between the two groups on the 7th,14th and 21st day (P<0.05) in the neonate rat lungs. There was no significant difference of HoxB5 mRNA expression which expressed at a high level between the experimental group and the control group on the 1st and 3rd day (P>0.05). And there was significant difference between the two groups on the 7th,14th and 21st day (P<0.05). In the experimental group, HoxB5 mRNA expression decreased gradually and there was significant difference between the two groups on the 7th,14th and 21st day (P<0.05)
Hybridization in situ results showed that HoxA5 mRNA was expressed mainly in alveolar epithelial cells, mesenchymal cells and vascular endothelial cells. Small amounts of them expressed tunica mucosa bronchiorum cells in the lung which expressed both in karyoplast and kytoplasm. HoxA5 mRNA was mainly located in alveolar septum cells and alveolar epithelial cells. With the alveolization development, its expression was more obvious in alveolar epithelial cells. In the control group, the level of HoxA5 mRNA was high and there was no significant difference between the 1st and 3rd day(p>0.05). Its expression was steady and kept on a high level after 7 days. Compared with the control group, HoxA5 mRNA expression in the experimental group began to decrease on the 7th day, it remained at a low level on the 21st day(P<0.05) while there was no significant difference between the 1st and 3rd day(p>0.05). HoxB5 mRNA was mainly expressed in alveolar epithelial cells, tunica mucosa bronchiorum cells and vascular endothelial cells, and small amounts of them expressed in mesenchymal cells of the neonate rats lung. With the alveolization development, HoxB5 mRNA was expressed more and more obviously in the alveolar septum, junction and cristae. In the control group, the level of HoxB5 mRNA expression was high and there was no significant difference between the 1st and 3rd day (P>0.05), it reached the peak on the 7th day, and its expression was steady and kept on a high level on the 14th day and 21st day. Compared with the control group, there was no significant difference of HoxB5 mRNA expression between the two groups on the 1st day (p> 0.05). HoxB5 mRNA expression began to decreas on the 3rd day, the difference of HoxB5 mRNA expression was significant between the two groups on the 7th,14th and 21st day (P<0.05)
Concludsion
1.Pathological findings charactered by the arrested alveolar development and deposited fibres in newborn rats after prolonged hyperoxia-exposed are consistent with those of CLD of prematurity in human beings.
2. SPC is expressed mainly in the typeⅡalveolar epithelial cells in the lung. AQP5 is expressed in the typeⅠalveolar epithelial cells in the lung. AQP5 is also expressed in the tunica mucosa bronchiorum and small vessels submucosa.
3. The number of AEC-Ⅱdecreased in early stage of CLD, then it would keep in a higher level,while the number of AEC-Ⅰdecreased gradually which was the changes of alveolar epithelial cells in the impaired lungs induced by hyperoxia.
4. AEC-Ⅱis the stem cells of AEC-Ⅰ, and so we suppose that there would be dysdifferentiation of AEC-Ⅱto AEC-Ⅰin the hyperoxia induced CLD.
5. HoxA5 and HoxB5 genes are expressed in the normal neonate rats. In the period of alveolization, the tendency of the two genes expressed amount is upgraded. Both of the two genes are expressed in alveolar epithelial cells especially in the alveolar septum junction and cristae.
6.The expression of HoxA5 and HoxB5 genes is abnormal in the impaired lungs induced by hyperoxia. With the extended exposure time, the amount of their expression deceased gradually.
7. At the early stage of hyperoxia induced lung injury alveolar epithelium is damaged and alveoli of lung reparation is blocked. HoxA5 and HoxB5 genes might regulate AEC-Ⅱdifferentiated to AEC-Ⅰ.
早产儿慢性肺疾病(chroniclungdisease,CLD)是早产儿长时间吸入高浓度氧或机械通气治疗或感染后最常见的和最严重的并发症。早产儿CLD作为早产儿肺透明膜病治疗的并发症在1967年首次被Northway描述,也称为支气管肺发育不良(bronchopulmonary dysplasia,BPD)。氧疗法是患有心肺疾病的早产儿最常应用的一种治疗手段。这一措施虽可改变患儿的低血氧状态、挽救患儿的生命,但长期吸入高浓度的氧气,可引起不同程度的肺损伤,严重者可发展为CLD。随着机械通气的广泛开展、早产儿管理技术的日益提高及肺表面活性物质的普遍应用,使早产儿,特别是超低出生体重儿(extremely low birth weight, ELBW)(出生体重<1000g存活率有了明显改善,但随之而来的是早产儿慢性肺疾病发生率的提高,国外已达30%-40%,国内也有上升趋势。由于尚缺乏有效的监控和防治手段,其中10%-15%CLD患儿因严重肺功能障碍而死于呼吸衰竭,存活者也需长期依赖氧气或机械通气治疗。
初期肺泡上皮细胞(alveolar epithelial cell,AEC)损伤和晚期肺纤维化是CLD主要病理特征。以往的研究侧重阐明CLD的AEC损伤(坏死或调亡)及其损伤后纤维组织替代AEC的病理过程及其可能机制,故目前多试图通过减轻AEC损伤程度或减少肺组织胶原沉积而达到有效防止CLD的目的,但效果均不理想,至今在CLD防治措施方面的研究进展缓慢。但恰是这种正常肺上皮重塑障碍后的不可逆肺纤维化,严重影响了患儿的呼吸功能。那么损伤后的肺泡上皮为何不能完全修复而被纤维组织替代呢?目前尚无答案。因此,我们设想如以研究高氧致CLD中AEC的修复及其调控机制为切入点,可能是解决肺上皮重塑障碍的重要突破口。
AEC包括Ⅰ型肺泡上皮细胞(typeⅠalveolar epithelial cell, AEC-Ⅰ)和Ⅱ型肺泡上皮细胞(typeⅡalveolar epithelial cell, AEC-Ⅱ),已证实AEC-Ⅰ为终末已分化细胞,无再生、增殖及分化能力；目前研究证实AEC-Ⅱ是AEC-Ⅰ的干细胞或祖细胞,如体外培养的AEC-Ⅱ,可很快失去其特有的标志SPC(surfactant proteinC),而表达AEC-Ⅰ的特有标志AQP5 (aquaporin5);用3H-TDR标记动物体内的AEC-Ⅱ,2天后在AEC-Ⅱ临近发现标记的AEC-Ⅰ等。由此可见,若肺上皮损伤后,AEC-Ⅰ无再生、修复能力,完全依赖AEC-Ⅱ的增殖、分化方能完成正常的肺上皮的重塑。
近年来,由于Hox基因(homeobox genes, Hox genes)在进化过程中的极大保守性和其在动物胚胎发育分化中的作用,使之成为当今世界生命科学最热门的研究领域之一。而有关Hox基因对正常肺组织发育及细胞分化调控作用的研究尚处起步阶段。多数文献阐明不同簇的Hox基因在胎肺发育的特定阶段、特定部位表达,对肺细胞系分化、成熟期重要作用,如HoxA5和HoxB5与支气管分枝形成,HoxB7、HoxB8和HoxB5与肺芽发育密切相关。有个别报道HoxB5异常表达与先天性肺隔离症、HoxA5过渡表达与原发性肺动脉高压、肺气肿发生有关。Hox基因对生后肺发育的调控机制目前尚不清楚,生后肺组织中仍存在Hox基因,其中表达最强的是HoxA5,,其次是HoxB5,且这两种基因主要定位于肺泡上皮细胞,而AEC-Ⅰ又为终末已分化细胞,故我们推测,高氧CLD时,能否因AEC-Ⅱ的HoxA5、HoxB5基因的表达降低而导致其向AEC-Ⅰ的分化障碍。
我们假设：HoxA5和HoxB5基因可能是AEC-Ⅱ向AEC-Ⅰ分化的主控基因之一,其表达异常导致了损伤AEC-Ⅰ的修复障碍,最终发生肺上皮再生受抑,纤维组织增生替代的病理结局。如该假设成立,不仅可以阐明在正常早产鼠肺发育及CLD发生、发展中HoxA5、HoxB5时空表达模式,更重要的是发现Hox同源盒基因对AEC-Ⅱ分化的调控作用,从而为进一步探索有效防治早产儿CLD的新途径奠定理论和实验基础。
实验材料与方法
一、动物模型
本实验是在中国医科大学盛京医院实验动物中心完成。足月新生Wistar大鼠(连同母鼠)被随机分为2组：A组即试验组、单纯高氧暴露；B组即对照组、正常空气吸入；将A组在生后24 h内置于玻璃氧舱中,持续输入氧气,维持在FiO20.85,每日两次监测氧浓度(美国OM-25ME型测氧仪监测),用钙石灰吸收CO2,使其浓度小于0.5%(美国Dapex气体分析仪),温度20℃-25℃,湿度50%-70%,每天开箱0.5h,称重,添加水及饲料,更换垫料,与空气组互换母鼠以避免母鼠因氧中毒而致护理能力降低；空气对照组置于空气中(Fi020.21),饲养条件与高氧组相同。
二、标本采集和处理
分别于实验开始后的第1、3、7、14、21天分别从每组中随机抽取8只新生鼠,腹腔注射5%水合氯醛6ml/kg麻醉后处死。立即打开胸腔,无菌条件下分离肺组织,取右肺各叶置于无Rnase的Eppendorf管中于-80℃冰箱中保存。取左肺置于0.1%DEPC的4%多聚甲醛(0.1MPBS, pH7.0-7.6)固定,常规脱水、石蜡包埋。
三、实验方法
1、动态观察各组新生大鼠的一般状态、监测体重。
2、病理形态学研究
(1)光镜观察：切片进行常规HE染色,光镜下动态观察肺组织的病理改变。
(2)肺泡面积/肺间隔面积(A/S)(美国Universal Ima-grog Porporation图像分析系统)。
(3)放射状肺泡计数(radical alveolar counts, RAC)。
(4)透射电镜观察AEC-Ⅱ超微结构改变。
3、采用免疫组化(Immonohistochemistry, IH)、RT-PCR检测肺组织AQP5、SPC的表达
4、两组肺组织HoxA5、HoxB5蛋白含量和基因表达的动态变化
(1)免疫组化方法检测肺组织HoxA5、HoxB5在肺组织中表达的强度和部位。
(2) RT-PCR技术检测肺组织HoxA5、HoxB5mRNA的表达。
(3)原位杂交(Hybridization in situ, ISH)检测表达HoxA5、HoxB5mRNA强度和部位。
(4)Western-blot (WB)方法检测肺组织HoxA5、HoxB5蛋白定量。
四、统计学分析
应用SPSS13.0统计软件进行统计学处理,数据以均值±标准差(x±s)表示,单因素多水平比较采用One Way ANOVA。两样本均数间比较采用Independent t test。相关分析采用Spearman分析。结果以P<0.05有意义。
结果
一、实验组和对照组新生大鼠一般状态比较
实验组新生鼠1d肤色红润,脱离氧气无呼吸困难,3d离氧后出现呼吸急促,一般在7d后出现少动、呼吸急促明显、皮肤苍白以及离氧后不同程度的发绀,随高氧暴露时间延长而逐渐加重,在14d后出现对氧的依赖。而对照组组则无以上异常表现。
实验组在高氧暴露7d开始,新生大鼠体重与对照组相比下降(P<0.05),随着高氧暴露时间的延长,体重差异逐渐明显(P<0.01)。
二、两组新生大鼠肺组织病理形态学变化的比较
1、肺组织的病理改变：
对照组1d的肺组织出现小而浅的原始肺泡,形态不规则,肺泡间隔较厚；3d时终末气腔大小降低,数量渐增多,肺泡间隔渐薄；7d-21d肺泡化进一步完善,到21d肺泡间隔变薄,肺泡次级隔明显增多,肺泡数量增多,形态较规则；实验组在高氧暴露1d后与对照组无明显差别。3d可见小血管扩张、充血,肺泡腔内或间隔少量出血,以中性粒细胞为主的渗出、间质内细胞增多。7d时炎症反应进一步加重,肺泡间隔水肿变宽,出现终末气腔扩张、肺泡融合、肺泡分隔减少等。14d-21d间隔增宽,小灶或多灶状实变,肺组织正常结构破坏,且终末气腔扩张更为明显、小肺泡数量更少,肺泡次级隔明显减少。
2、A/S和RAC动态变化：
生后1d和3d,两组A/S和RAC均无差异(p>0.05)。7d,14d,实验组的A/S、RAC明显低于对照组(p<0.01),A/S高于对照组(p<0.01),21d时A/S升至最高和RAC降至最低(p<0.001)。
3、肺泡Ⅱ型上皮细胞超微结构改变
正常AEC-Ⅱ的表面有大量微绒毛,细胞结构完整,胞核呈椭圆形,核膜完整,染色质分布均匀；胞浆内含有线粒体和大量板层小体。吸高氧后1d,即AEC-Ⅱ的少数线粒体肿胀；3d时LB开始有排空现象；7d时细胞胞膜上微绒毛减少,胞核尚完整,染色质以异染色质为主,线粒体肿胀和板层小体空泡化；14d时,游离端大量微绒毛突出细胞表面并少量脱落,线粒体嵴断裂,部分溶解,半层小体基本排空；21d细胞核固缩、溶解,线粒体、板层小体、微绒毛结构均消失。
三、高氧致慢性肺疾病新生鼠肺泡上皮细胞SPC和AQP5表达及动态变化
1、高氧后新生大鼠肺组织SPC、AQP5蛋白表达的动态变化
免疫组化结果示：SPC为Ⅱ型肺泡上皮细胞标志蛋白,主要在胞浆中表达。实验组3d明显低于对照组(P<0.05),1d、7d无明显差异,14d、21d明显高于对照组(P<0.05)；AQP5为Ⅰ型肺泡上皮细胞的标志蛋白,胞浆、胞膜可见棕黄色染色。其表达1d、3d两组无明显差异,7d、14、21d实验组均低于对照组,差异显著(P<0.05)。
2、高氧后新生大鼠肺组织SPC、AQP5mRNA表达动态变化
SPCmRNA的表达不呈直线改变,实验组与对照组比较,1d两组间无明显差异,3d时实验组明显减少有显著差异,7d、14d、21d时其表达有明显增多,且相对量多于对照组,两组间差异不显著(P>0.05)。AQP5mRNA表达量实验组逐渐减少,14d、21d变化减缓；对照组逐渐增加且趋势平缓。两组间除1d外其他各组均存在显著差异(P<0.05)。
四、高氧致CLD新生大鼠肺组织HoxA5和HoxB5基因表达动态变化的实验研究
1、高氧后新生大鼠肺组织HoxA5、HoxB5蛋白表达的动态变化
免疫组化结果显示HoxA5在新生鼠肺组织中主要表达肺泡上皮细胞、肺间质细胞、血管内皮细胞,少量表达于支气管粘膜细胞。随着肺泡化的发生,其在肺泡上皮细胞表达增多趋势更明显。对照组1d、3d表达量均较多且无明显差别(P>0.05),7d后平稳表达并且维持在较高水平。实验组,1d、3d与对照组表达量无明显差异(P>0.05),7d开始表达量逐渐下降,14d表达最低,21d表达仍低于对照组(P<0.05)。HoxB5在新生鼠肺组织中主要表达于肺泡上皮细胞、支气管粘膜细胞、血管粘膜及粘膜下,肺间质亦有少量表达。随着肺泡化的发生,其在肺泡间隔及肺泡嵴上的表达也越明显。对照组1d、3d表达量均较多且无明显差别,7d表达达高峰,14d、21d平稳表达并且维持在较高水平。实验组,1d、3d与对照组表达量无明显差异(p>0.05),7d开始表达量逐渐下降,7d、14d、21d与对照组相比差异显著(P<0.05)。
Western结果与免疫组化结果基本一致,对照组HoxA5蛋白1d、3d表达较多,7d、14d、21d表达增多。实验组HoxA5蛋白1d、3d表达较多,7d、14d、21d表达减少且与对照组存在显著差异(P<0.05)。HoxB5蛋白1d、3d表达较多,7d、14d、21d表达增多。实验组HoxB5蛋白1d、3d表达较多,7d、14d、21d表达逐渐减少且与对照组存在显著差异(P<0.05),21d时其蛋白表达基本为阴性。
2、高氧后新生大鼠肺组织HoxA5、HoxB5mRNA表达的动态变化
RT-PCR结果显示：对照组HoxA5mRNA1d、3d表达较多,7d、14d、21d表达增多。实验组HoxA5mRNAld、3d表达较多,7d、14d、21d表达逐渐减少且与对照组存在显著差异(P<0.05)。HoxB5mRNA在新生鼠肺组织中表达,实验组与对照组之间1d、3d无明显差异(P>0.05),表达较多,而7d、14d、21d差异显著(P<0.05)。实验组7d之后HoxB5mRNA表达逐渐减少,至21d时表达已极其微弱,对照组表达的量逐渐增多,14d后表达平缓,7d、14d、21d两组间存在显著差异(P<0.05)。
原位杂交结果：HoxA5 mRNA在新生鼠肺组织中主要表达于肺泡上皮细胞、肺间质细胞、血管上皮细胞,支气管粘膜上皮细胞也有少量表达。在细胞核和／胞浆中均有表达,1d、3d时肺泡间隔细胞和肺泡上皮细胞表达均较多,随着肺泡化的发生,其在肺泡上皮细胞表达增多趋势更明显,实验组3d细胞核表达量较多。对照组1d、3d表达量均较多且无明显差别(P>0.05),7d后平稳表达并且维持在较高水平。实验组,1d、3d与对照组表达量无明显差异(P>0.05),7d开始表达量逐渐下降,21d表达很少,7d、14d、21d两组差异显著(P<0.05)。HoxB5 mRNA在新生鼠肺组织中主要表达于肺泡上皮细胞、支气管粘膜细胞、血管粘膜及粘膜下,肺间质亦有少量表达。随着肺泡化的发生,其在肺泡间隔、肺泡连接处及肺泡嵴上的表达也越明显。对照组1d、3d表达量均较多且无明显差别(P>0.05),7d表达达高峰,14d、21d平稳表达并且维持在较高水平。实验组,1d与对照组表达量无明显差异(P>0.05),3d时表达已开始减少,7d、14d、21d表达水平均较低,与对照组差异明显(P<0.05)。
结论
1、新生大鼠持续高浓度氧气暴露,可导致新生大鼠肺组织出现肺泡发育障碍和肺纤维组织增生等病理形态学改变,符合早产儿CLD的发生发展特点和解剖学改变。
2、高氧肺损伤肺泡发育停滞或者肺泡损伤后修复障碍在新生鼠CLD早期即有明显表现,随着新生鼠高氧暴露时间的延长这一特点更加明显。
3、新生大鼠肺组织SPC、AQP5主要表达于肺泡上皮细胞。AQP5在支气管粘膜和小血管粘膜下层亦有表达。高氧7d后SPC表达量增多但是表达较紊乱,肺泡间隔细胞表达较多。
4、根据肺泡上皮细胞特异性标志物的变化,高氧致肺损伤,肺泡上皮细胞改变可能为：AEC-Ⅱ早期减少,随后增加并维持在较高水平；AEC-Ⅰ进行性减少。
5、既然AEC-Ⅱ是AEC-Ⅰ的干细胞,推测高氧致新生鼠CLD肺泡损伤时可能存在AEC-Ⅱ向AEC-Ⅰ的分化障碍。
6、HoxA5和HoxB5基因在正常新生鼠肺组织中表达,在肺泡形成期,表达量呈上升趋势。两个基因均在肺泡上皮中表达,在肺泡嵴、肺泡拐角、肺泡连接处表达更明显。
7、高氧致新生鼠肺损伤导致HoxA5、HoxB5基因表达异常,且随着暴露时间的延长,其表达量渐低。
8、高氧致肺损伤早期肺泡上皮损伤,肺泡修复障碍,HoxA5、HoxB5基因可能调控AEC-Ⅱ转化为AEC-Ⅰ。
Introduction
Chronic lung disease (CLD) of prematurity is the most frequent and severe complication that is caused by inhaling concentration oxygen, mechanical ventilation therapy or lung infection. Bronchopulmonary dysplasia (BPD) is one of chronic lung diseases first described in 1967 as a complication of therapy for premature infants with hyaline membrane disease. At the same time treatment with high concentration of oxygen was thought to be a major contributor to its development. Since administration of supplemental oxygen as one of supportive therapies elevates oxygen concentrations, that may cause pulmonary oxygen toxicity, and even develop to CLD. Improvements in respiratory care and management had allowed infants of more than 30 wk of gestation to survive their respiratory distress syndrome, even before the widespread useof exogenous surfactant therapy. This had led to an increased survival of very immature infants (BW<1000g), who were at most risk of developing bronchopulmonary dysplasia.(reaching 30%-40% abroad, upgrading tendency domestic). In total about 10%-15% CLD infants died with respiratory failure due to lung functional disturbance, because of deficiency of effect monitoring and treating means. Consequently the survival have to rely on oxygen or mechanical ventilation for a long term.
Pulmonary fibrosis has happened followed with alveolar epithelial cell (AEC) damage which is the main pathology characteristic. Formerly, lots of studies tried to illuminate the mechanism of the alveolar epithelial cell impairment (necrosis or apoptosis) and the pathological process of fibrous tissue substituting AEC. In order to prevent CLD many experiments have been done to relieve the degree of AEC injury and the amount of collagen deposition. Unfortunately there has no effective measures.So far the development of the research about CLD treatment is still on the slow pace. It is just the inreversible pulmonary fibrosis after the normal alveolar epithelial cell defeated in remodeling that impacts the infants'respiratory function. Why the damaged AEC couldn't be repaired but substituted by fibrous tissue? There is no answer for it. So we assume that studying the reparation of AEC and the mechanism of its regulation might be the break to resolve the problem about the normal alveolar epithelial cell defeated in remodeling.
The alveolar epithelial cells consist of typeⅠ(AEC-Ⅰ) and typeⅡ(AEC-Ⅱ)cells. It has been confirmed that typeⅠalveolar(epithelial)cells (AEC-Ⅰ) are the noble cells which can't be regenerated, proliferated and differentiated. Meanwhile typeⅡalveolar (epithelial) cells (AEC-Ⅱ) are the main stem cells in the lung. If AEC-Ⅱare cultured in vitro they will lose their specific marker SPC while expressing aquaporin5 (AQP5) which is the specific marker of AEC-Ⅰ. After 3H-TDR tagged animal endosomatic AEC-Ⅱfor 2 days, the labelled AEC-I would be found near the AEC-Ⅱ. It can be seen that if the alveolar epithelial cells are damaged it would depend on AEC-Ⅱto proliferate and differentiate to accomplish the epithelial remodeling as AEC-I hasn't the ability of regeneration and reparation.
For the past few years the function of homeobox genes (Hox genes) in embryonic development and differentiation has been one of the hot topics all around the world because of Hox genes'conservatism in evolutionary process. The study about hox genes in normal lung development and its functionary in pneumocyte differentiation is in early stage. Different Hox genes are expressed in different regions during embryonic lung development which are important to lung cell lines differentiation and maturation. For example, there is an intimate correlation between hoxa5 and hoxb5 and branching morphogenesis, and so is hoxb7, hoxb8 and hoxb5 and lung bud development. Several reports have described that the normal expression of hoxb5 is concerned with congenital pulmonary sequestration and the excessive expression of hoxb5 is concerned with primary pulmonary hypertension and emphysema. The mechanism of the regulation of hox genes expression in the post-lung has not been clear. Hox genes are also expressed in the neonatal lung,. The expression of hoxa5 is the most intensive followed by hoxb5. The two genes are mainly located in alveolar epithelial cells, and AEC-Ⅰis the ultimate differentiated cell.So we assume that the decrease of expression of hoxa5 and hoxb5 might be the cause of dysdifferentiation of AEC-Ⅱto AEC-Ⅰin the hyperoxia induced CLD.
We have a hypothesis:hoxa5 and hoxb5 may be two of the chief genes that control AEC-Ⅱdifferentiated to AEC-Ⅰ.The abnormal expression of them leads to failure of the injured AEC-Ⅰreparation, and results in the ultimate pathology of restrained reepithlialization at last and fibrous tissue substituting the epithelium. If the hypothesis was correct we could illuminate the time-space model of hoxa5 and hoxb5 genes'expression in the normal and CLD rats'lungs but also we could find out that hox genes might play a role in the regulation of the AEC-Ⅱdifferentiation. Thus it will provide experimental and theoretical basis to accomplish the pathogenesis and preventive and therapeutic methods of CLD in premature.
Materials and Methods
ⅠAnimal model
The present study was performed in accordance with the guidelines provided by the Experimental Animal Laboratory of Shengjing Hospital China Medical University. Within 24 hours after birth, pups were randomly redistributed to the newly delivered mothers. Term neonatal Wistar rats were divided randomly into two groups.A gruop is an experimental group,which was exposed to oxygen. B group is AIR group that served as controls and were kept in air and not subject to hyperoxia. The pups of hyperoxia exposure groups were contained in glass chambers in which the oxygen was infused continuously to achieve 85% oxygen concentration which was monitored with an oxygen monitor(OM-25ME, USA) twice daily. CO2 was removed by soda lime absorption to keep CO2 levels below 0.5%(Dapex Gas Monitor, USA). Temperature and Humidity was maintained at 20℃-25℃and 50%-70%respectively. We opened the chambers for 0.5h to exchange nursing mothers between air and O2-exposed groups every 24 hours to prevent maternal O2 toxicity and eliminate maternal effects between groups. Meanwhile change water, add food, clean dirty cages and record body weight. All animals were raised in the same room and all other conditions were the same.
ⅡTissue preparation
On postnatal 1d,3d 7d,14d,21d. Each group of hyperoxia as well as air rats were anesthetized by intraperitoneal injection of 5% chloral hydrate (6ml/kg), and put to death. The thorax was opened and the lungs were resected. The right lung tissues were put in the Rnase-free Ependorf tubes, and immediately frozen in liquid N2 for mRNA, Enzyme linked immunoadsorbent assay(ELISA), and so on. The left lung tissues were fixed in 4%formaldehydum polymerisatum which contained 0.1% DEPC(0.1MPBS, pH7.0-7.6), then were dehydrated, embedded in wax within 24 hours.
ⅢExperiment methods
1.The appearances and weight were monitored daily in each experiment groups.
2.The changes of the lung pathology
(1)For lung pathology assessment, lung sections from the left lobes were stained with HE (hematoxylin and eosin).
(2)Ratio of alveolar and septa area(A/S).
(3)Radical alveolar counts(RAC).
(4)Electronic microscopy:the observation of the ultrastructures of AECII.
3.Using Immonohistochemistry and Reverse transcription polymerase chain reaction(RT-PCR) to detect protein and mRNA of AQP5 and SPC expression in the lung.
4.The measurement of protein and gene level of HoxA5 and HoxB5 dynamic expression in the lung tissue
(1)Immonohistochemistry:the detection of intensity and position of the protein expression in lung tissue about HoxA5 and HoxB5
(2)RT-PCR:the detection at mRNA level of HoxA5 and HoxB5 in the lung tissue.
(3)Hybridization in situ:the detection of intensity and position of the mRNA expression in lung tissue about HoxA5 and HoxB5
(4)Western-blot:the detiction at the protein level of HoxA5 and HoxB5 in the lung tissue of neonates rats.
IV Statistical analysis
SPSS versionl3.0 was used to perform statistical analysis with all data expressed by (mean±SD). Statistically significant differences in the mean values among multiple groups were analyzed using One Way ANOVA analysis.The mean value of two groups were analyzed with Independent-Samples T Test. Correlation between two variables was analyzed with Spearman analysis. Statistically significant differences set P<0.05.
Results
ⅠGeneral status of rats in experimental groups and control groups
The hyperoxia rats were didn't present dyspnea after one day of oxygen exposure. Tachypnea was observed on the 3rd day after they were detached from oxygen supplementation. After the 7th day of oxygen exposure they began to appear fatigue, pale, and to present tachypnea and cyanosis for different degrees. After 14 days some even had to rely on high oxygen and become worse with the time gone. The control groups didn't have the appearances above.
At the beginning there was no difference in average birth weight between the two groups. From 7 days of oxygen exposure, weights of the experimental groups showed a significant decrease compared with control groups at the sams time period(P<0.05).
ⅡComparision of lung pathomorphism of the two neonatal rats' groups
1. The pathological changes of the lung tissue:In the control groups,on the 1st day of the experiment, it was observed that the alveolar structure was irregular, terminal air space size was rather small, and the alveolar septum was thick. On the 3rd day, the alveolar structure of each group was more regular, the size of alveolus was equal, and alveolar septum was thinner, but in the experimental group, there were a few inflammatory cells exuded out, and blooding interstitial cells increased. On the 7th day the alveolar size was equal in room air rats, while the terminal air space size of the oxygen-exposed rats became large, and there was inflammatory response and more interstitial cells. On 14th day and 21st day, air groups continued to develop alveolization, the alveolar became more regular, the size of alveolus was equal and the alveolar septum was thinner; but in O2 groups, the terminal air space size grew significantly large, secondary septum decreased, the quantity of alveolar reduced with alveolus fusion, interstitial cells increased, and alveolar septa was thicker. On the whole the severity of pathological changes was parallel to the duration of exposure.
2. The dynamic changes of ratio of alveolar and septa area(A/S) and Radical alveolar counts(RAC):No significant differences about A/S and RAC could be seen between experimental groups and control groups on day 1-3(p>0.05). But there was a significant difference between the experimental groups and the control groups on the 7th and 14th day(p<0.01). On the 21st day, A/S increased to the highest level and RAC decreased to the lowest level.
3. Ultrastructure changes of AECⅡ:There were large normal microvilli (Mv) on the surface of AECⅡ, the structure of the cell was in integrity, the nucleus was ellipse, nuclear membrane was complete, and chromoplasm was distributed evenly; There were mitochondrias(Mi) and large number lamellar bodies(LB) in the intracytoplasm. After exposure hyperoxia for one day, some mitochondrias were swelling. LB began to be emptied on the 3rd day; and on the 7th day the number of Mv was decreased,the main chromoplasm was heterochromatin, Mi swelled and LB was vacuolization, but the nucleus was complete. On the 14th day large number of microvilli outpocketed from the membrane and several of them ablated. Mitochondrial cristae broke and some of them dissolved. On the 21st day there was karyopyknosis and cell nucleus dissolved,and d Mi, LB and Mv disappeared.
Ⅲ.The dynamic changes of SPC and AQP5 expression in the lung tissue of hyperoxia induced CLD
1. The dynamic changes of SPC and AQP5 protein expression in the neonate rat lung after being exposed in hyperoxia
Immuonhistochemical studies shows SPC is an identification marker of AECⅡ, which is expressed in kytoplasm of the typeⅡalveolar epithelial cells in the lung. Compared with the control group, SPC expression in the experimental group decreased significantly on the 3rd day (P<0.05) while it increased obviously on the 14th and 21st day (P<0.05),but there was no difference on the 1st and 7th day. AQP5 is an identification marker of AECI, which is expressed in kytoplasm and cell membrane of the typeⅠalveolar epithelial cells in the lung. On the 1st and 3rd day, there was no difference of its expression, but on the 7th,14th and 21st day its expression in the experimental group decreased significantly (P<0.05)
2. The dynamic changes of SPC and AQP5 mRNA expression in the neonate rat lung after being exposed in hyperoxia
The expression tendency of SPC mRNA was not in a straight line.Compared with the control group, SPC mRNA expression in the experimental group decreased significantly on the 3rd day (P<0.05),while it increased obviously on the 7th,14th and 21st day (P<0.05) and its relative amount is more than that in the control group, but there was no difference on the 1st day. AQP5 mRNA expression in the experimental group decreased gradually, and its change slowed down on the 14th and 21st day. The level of AQP5mRNA in the control group increased gradually and its tendency was gentle. There was significant difference between the two groups except the 1st day.
IV Empirical study of the dynamic changes of HoxA5 and HoxB5 genes expression in the lung tissue of hyperoxia induced CLD
1. The dynamic changes of HoxA5 and HoxB5 protein expression in the neonate rat lung after being exposed in hyperoxia
Immuonhistochemical results showed that HoxA5 protein was mainly expressed in alveolar epithelial cells, mesenchymal cells and vascular endothelial cells, and small amounts of them expressed tunica mucosa bronchiorum cells in the lung. With the alveolization development, the tendency of HoxA5 protein expression was more obvious in alveolar epithelial cells. In the control group, the level of HoxA5 protein expression was high and there was no significant difference from the 1st to 3rd day(p> 0.05), and its expression was steady and kept on a high level after 7 days. Compared with the control group, HoxA5 protein expression in the experimental group began to decrease on the 7th day and got to the lowest level on the 14th day, and it remained on a low level on the 21st day (P<0.05),while there was no significant difference between the 1st and 3rd day(p>0.05). HoxB5 protein was mainly expressed in alveolar epithelial cells, tunica mucosa bronchiorum cells and vascular endothelial cells, and small amounts of them expressed in mesenchymal cells of the neonate rat lungs. With the alveolization development, more HoxB5 protein was obviously expressed in the alveolar septum and cristae. In the control group, the level of HoxB5 protein expression was high and there was no significant difference between the 1st and 3rd day, it reached the peak on the 7th day, and its expression was steady and kept on a high level on the 14th day and 21st day. Compared with the control group, there was no significant difference of HoxB5 protein expression between the 1st and 3rd day(p>0.05). HoxB5 protein expression decreased gradually on the 7th day, and the difference of HoxB5 protein expression was significant between the two groups on the 7th,14th and 21st day (P<0.05)
Western blotting got the similar results as those in imunohistochemistry. In the control group, the level of HoxA5 protein expression was high and there was no significant difference between the 1st and 3rd day(p>0.05), HoxA5 protein expression increased on the 7th,14th and 21st day. In the experimental group, the level of HoxA5 protein expression was high on the 1st and 3rd day, HoxA5 protein expression decreased and there was significant difference between the two groups on the 7th,14th and 21st day (P<0.05). In the control group, the level of HoxB5 protein expression was high on the 1st and 3rd day, HoxB5 protein expression increased on the 7th,14th and 21st day. In the experimental group, the level of HoxB5 protein expression was high on the 1st and 3rd day, HoxB5 protein expression decreased gradually and there was significant difference between the two groups on the 7th,14th and 21st day (P<0.05), and the protein expression of HoxB5 was almost negative on the 21st day.
2 The dynamic changes of HoxA5 and HoxB5 mRNA expression in the neonate rat lung after being exposed in hyperoxia
RT-PCR results showed:In the control group, the level of HoxA5 mRNA expression was high on the 1st and 3 rd day, and HoxA5 mRNA expression increased on the 7th,14th and 21st day. In the experimental group, the level of HoxA5 mRNA expression was high on the 1st and 3rd day, HoxA5 mRNA expression decreased gradually and there was significant difference between the two groups on the 7th,14th and 21st day (P<0.05) in the neonate rat lungs. There was no significant difference of HoxB5 mRNA expression which expressed at a high level between the experimental group and the control group on the 1st and 3rd day (P>0.05). And there was significant difference between the two groups on the 7th,14th and 21st day (P<0.05). In the experimental group, HoxB5 mRNA expression decreased gradually and there was significant difference between the two groups on the 7th,14th and 21st day (P<0.05)
Hybridization in situ results showed that HoxA5 mRNA was expressed mainly in alveolar epithelial cells, mesenchymal cells and vascular endothelial cells. Small amounts of them expressed tunica mucosa bronchiorum cells in the lung which expressed both in karyoplast and kytoplasm. HoxA5 mRNA was mainly located in alveolar septum cells and alveolar epithelial cells. With the alveolization development, its expression was more obvious in alveolar epithelial cells. In the control group, the level of HoxA5 mRNA was high and there was no significant difference between the 1st and 3rd day(p>0.05). Its expression was steady and kept on a high level after 7 days. Compared with the control group, HoxA5 mRNA expression in the experimental group began to decrease on the 7th day, it remained at a low level on the 21st day(P<0.05) while there was no significant difference between the 1st and 3rd day(p>0.05). HoxB5 mRNA was mainly expressed in alveolar epithelial cells, tunica mucosa bronchiorum cells and vascular endothelial cells, and small amounts of them expressed in mesenchymal cells of the neonate rats lung. With the alveolization development, HoxB5 mRNA was expressed more and more obviously in the alveolar septum, junction and cristae. In the control group, the level of HoxB5 mRNA expression was high and there was no significant difference between the 1st and 3rd day (P>0.05), it reached the peak on the 7th day, and its expression was steady and kept on a high level on the 14th day and 21st day. Compared with the control group, there was no significant difference of HoxB5 mRNA expression between the two groups on the 1st day (p> 0.05). HoxB5 mRNA expression began to decreas on the 3rd day, the difference of HoxB5 mRNA expression was significant between the two groups on the 7th,14th and 21st day (P<0.05)
Concludsion
1.Pathological findings charactered by the arrested alveolar development and deposited fibres in newborn rats after prolonged hyperoxia-exposed are consistent with those of CLD of prematurity in human beings.
2. SPC is expressed mainly in the typeⅡalveolar epithelial cells in the lung. AQP5 is expressed in the typeⅠalveolar epithelial cells in the lung. AQP5 is also expressed in the tunica mucosa bronchiorum and small vessels submucosa.
3. The number of AEC-Ⅱdecreased in early stage of CLD, then it would keep in a higher level,while the number of AEC-Ⅰdecreased gradually which was the changes of alveolar epithelial cells in the impaired lungs induced by hyperoxia.
4. AEC-Ⅱis the stem cells of AEC-Ⅰ, and so we suppose that there would be dysdifferentiation of AEC-Ⅱto AEC-Ⅰin the hyperoxia induced CLD.
5. HoxA5 and HoxB5 genes are expressed in the normal neonate rats. In the period of alveolization, the tendency of the two genes expressed amount is upgraded. Both of the two genes are expressed in alveolar epithelial cells especially in the alveolar septum junction and cristae.
6.The expression of HoxA5 and HoxB5 genes is abnormal in the impaired lungs induced by hyperoxia. With the extended exposure time, the amount of their expression deceased gradually.
7. At the early stage of hyperoxia induced lung injury alveolar epithelium is damaged and alveoli of lung reparation is blocked. HoxA5 and HoxB5 genes might regulate AEC-Ⅱdifferentiated to AEC-Ⅰ.
引文
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