β-catenin在高氧致新生鼠BPD中的作用及其机制的研究
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摘要
前言
支气管肺发育不良(Bronchopulmonary dysplasia,BPD)是采用机械通气或氧气治疗早产儿严重心肺疾病后常见的并发症,是威胁早产儿健康和生命质量的首要疾病。随着围产医学的不断发展,极低出生体重儿的成活率明显提高,BPD的发病率也在不断上升,其病理特点由过去显著的肺间质纤维化转变为以肺泡发育阻滞为突出特征。
BPD的发病机理包括:肺脏发育极不成熟,外界损伤以及损伤后修复机制的破坏。故此,BPD的发生可简单理解为在正常肺发育过程中,外界损伤导致的肺发育受阻和异常修复。以往的研究多着重于对肺组织损伤机制的研究,已证实氧毒性和机械通气介导的损伤、炎症反应在BPD的发生过程中具有重要作用,但抗炎、抗氧化的治疗对BPD却没有取得确切的疗效。随着对BPD认识的不断深入,对于即参与正常肺发育又调控肺组织损伤修复机制的研究,成为研究BPD发病机制的新视野。已证实肺发育过程中,位于肺泡分隔顶端的弹性蛋白不断向前延伸发展,是肺泡逐渐形成的物质基础;而细胞外基质的异常分布、沉积则是纤维化发生的直接原因。与两者的发生均密切相关的肺成纤维细胞(lung fibroblast,LF)的作用日益受到人们的关注。LF的增殖活化是其执行生物学功能的基础,对调节其增殖活化的信号通路的认识将有助于我们进一步理解LF的功能及其在BPD发生中的意义。
WNT信号通路是近年来才逐渐被人们认识到的一类控制细胞生长、增殖、传递细胞间相互调控信息的信号通路。其中WNT/β-catenin信号通路作为经典的WNT信号通路,已成为发育生物学及肿瘤学研究的热点。大量的研究证实,此信号通路活化的关键在于胞浆中是否存在结构稳定的β-catenin,以及其向胞核的转位。已有研究表明,早期肺发育(即支气管树形成)过程中WNT/β-catenin信号通路发挥了重要作用;且在对成人肺纤维化疾病的研究中发现WNT/β-catenin信号通路可能起着关键性的作用。但在肺泡化过程中WNT/β-catenin信号通路是否存在,其作用如何,在肺泡化障碍及肺纤维化的早产儿BPD中有何作用?到目前为止,鲜有报道。既然WNT/β-catenin信号通路参与调节肺发育及肺纤维化,且与细胞增殖分化有关,那么其在BPD中的作用是否通过促进LF增殖活化来实现?
本研究拟从组织和细胞水平,观察WNT/β-catenin信号通路关键因子β-catenin及其下游基因TCF-1/LEF-1的活化情况,探讨WNT/β-catenin信号通路在BPD中的作用及其机制,完善BPD的发病机制,为BPD的预防和治疗提供一个新思路。
实验材料与方法
一、动物模型
新生Wistar大鼠生后12h之内随机分为高氧组(A组)和空气组(B组)。A组置于玻璃氧箱中,持续输入氧气,FiO_2=0.85±0.02(美国OM-25ME型测氧仪监测)。用钠石灰吸收CO_2,使其浓度＜0.5%(Dapex气体分析仪),温度22～25℃,湿度60%～70%。每天定时开箱15min,添水、饲料及更换垫料,并与空气组交换母鼠以免因氧中毒而致喂养能力下降。B组置于空气中(FiO_2=0.21),余控制因素同A组。
二、标本的采集和处理
每组分别于实验后3、7、14d随机选取8只称重,5%哥拉腹腔注射(0.6ml/100mg)麻醉后,分离气管,打开胸腔,暴露心肺,经气管缓慢注入4%多聚甲醛,然后置于4%多聚甲醛中,4℃过夜,石蜡包埋,制作4μm组织切片,用于肺形态学观察、免疫组织化学检测。或于打开胸腔后,剪开左心耳,经右心室插入套管至肺动脉,注入10ml生理盐水洗净肺内残血,收集肺组织,置于无RNase的Eppendorf管中,液氮速冻,—80℃冰箱保存,用于RT-PCR及Western-blot检测。
三、细胞培养
动物模型建立后,分别于实验第3、7、14天取每组2-3只动物进行肺组织LF原代培养,取传2-3代细胞进行检测。
四、实验方法
1、组织水平
(1)动态观察各组新生大鼠的一般状态、监测体重。
(2)肺形态学观察:①H&E染色;②弹性蛋白染色;③Masson染色。
(3)免疫组织化学技术:α-平滑肌动蛋白(α-smooth muscle actin,α-SMA)、β-catenin蛋白检测。
(4)Western-blot法:检测肺组织β-catenin蛋白表达。
(5)RT-PCR法:检测肺组织α-表达。
2、细胞水平
(1)细胞培养及鉴定,MTT法检测细胞增殖情况。
(2)免疫细胞化学技术:α-平滑肌动蛋白(α-smooth muscle actin,α-SMA)、β-catenin、波形蛋白检测。
(3)免疫荧光:波形蛋白染色进行细胞鉴定。
(4)Western-blot法:检测细胞β-catenin蛋白表达。
(5)RT-PCR法:检测肺LF中β-catenin、TCF-1、LEF-1表达。
五、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((?)+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 ftest。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
A组新生鼠3d时肤色尚红润,脱离氧气后呼吸稍促,7d时出现少动、皮肤苍白以及离氧后呼吸频率稍快伴不同程度的发绀,随高氧暴露时间延长而逐渐加重,在14d时反应差,不活泼,体毛涩、无光泽,时有头颤;离氧后呼吸增快、出现呼吸困难、头颤明显,口周发绀。恢复氧供后可缓解。而B组始终反应较好,肤色红润,体重平稳增长,14天时睁眼。
A组在高氧暴露7d开始,体重与B组相比下降(p＜0.05),随着高氧暴露时间的延长,体重差异更加显著(p＜0.01)。
二、肺形态学改变
1、肺组织病理变化
A组3d肺泡结构紊乱,间隔少量炎性细胞浸润;7d间隔有大量炎细胞浸润,肺泡腔有渗出,肺灶状出血;14d肺泡数量明显减少,形成肺大泡,间隔增厚,肺间质胶原沉积。B组14d肺泡大小均匀一致,肺泡间隔较薄。
2、弹性蛋白染色
结果显示B组7天、14天时弹性蛋白主要表达于次级隔顶端,呈“蘑菇”样改变。A组则主要表达于肺间质。
3、Masson染色
结果显示B组肺泡间隔较薄,少量蓝色胶原沉积,A组14d蓝色胶原呈条索状或呈束状沉积于增厚的肺间质内。
三、细胞鉴定及细胞增殖测定
按Kelleher等建立的方法分离、培养LF,并进行鉴定:1、形态学鉴定:倒置显微镜下LF呈梭形,有较长的突起,胞核呈卵圆形,位于细胞中央,呈放射状或栅栏状排列;2、免疫荧光:波形蛋白表达于胞浆,绿色荧光为波形蛋白阳性染色;3、免疫细胞化学染色:胞浆内棕黄色颗粒为阳性染色。
MTT结果显示:A组LF生长较B组明显增加,实验各时间点均具有显著差异(p＜0.01)。
四、α-SMA蛋白在肺组织及LF中表达的动态变化
α-SMA主要表达于MF及血管平滑肌细胞。B组α-SMA 7天、14天时主要表达于次级隔顶端,与弹性蛋白表达部位基本一致,间质少有表达。A组7dα-SMA表达开始增加(p＜0.01),表达部位主要在间质,14d在上述部位表达明显增强(p＜0.01),与胶原蛋白表达一致性。在培养的LF中,胞浆中均存在α-SMA的阳性表达(＞95%),表达强度随高氧暴露时间延长而增强,7d、14d与B组存在显著性差异(p＜0.01)。
五、β-catenin在肺组织及LF中的表达
1、β-catenin蛋白在肺组织中的动态表达
免疫组化结果显示:B组3天肺组织支气管上皮细胞、部分肺泡上皮细胞、间质细胞胞核内均出现β-catenin表达,随着生后日龄的增加,在次级隔表达明显增多。A组自吸氧3d开始在上述细胞胞核中出现高表达,与B组具有显著性差异(p＜0.05),7d、14d仍呈高表达(p＜0.01),间质细胞核中表达明显增多。
Western-blot结果与免疫组化结果基本一致,两组β-catenin表达随日龄增长均逐渐增加,A组增加更加显著。实验第3天始,两组β-catenin表达就出现显著性差异(p＜0.05),7天及14天差异更为显著(p＜0.01)。
2、β-catenin基因在肺组织中的动态表达
RT-PCR结果显示:两组β-catenin基因表达随日龄增长逐渐增加,A组基因表达显著,其变化规律与蛋白表达趋势基本一致。两组内各时间点差异显著(p＜0.05)。
3、β-catenin蛋白在LF中的动态表达
免疫细胞化学结果显示:两组β-catenin均表达于细胞核内。
Western-blot结果显示:两组细胞β-catenin表达随日龄增长逐渐增加,A组增加更加显著,其变化规律与组织水平表达趋势基本一致。
4、β-catenin基因在LF中的动态表达
RT-PCR结果显示:两组β-catenin基因表达随日龄增长逐渐增加,A组基因表达增长显著,其变化规律与蛋白表达趋势基本一致。两组内各时间点差异显著(p＜0.05)。
六、TCF-1、LEF-1 mRNA表达的变化
1、TCF-1/LEF-1 mRNA在肺组织中的表达变化
RT-PCR结果显示:LEF-1 mRNA在A组自吸氧3d开始表达明显增强(p＜0.01),7d、14d仍呈高表达,与B组有明显差异(p＜0.05)。两组TCF-1 mRNA表达无明显差异(p＞0.05)。
2、TCF-1/LEF-1 mRNA在LF中的表达变化
RT-PCR结果显示:两组细胞中TCF-1/LEF-1 mRNA表达与组织水平变化趋势一致。随日龄增长逐渐增加,LEF-1 mRNA在A组3d表达增强(p＜0.01),7d、14d仍呈高表达,两组表达具有明显差异(p＜0.05),而两组TCF-1 mRNA在实验各时点表达无明显差异(p＞0.05)。
结论
1、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BPD的发生发展特点和病理学改变。
2、肺肌成纤维细胞的异常分布(未到达肺泡分隔顶端而沉积于间质)可能是BPD肺泡发育障碍的机制之一。
3、肺肌成纤维细胞沉积于间质并过度表达,与BPD肺泡损伤后修复障碍密切相关。肺肌成纤维细胞在BPD中的异常分布、表达可能是BPD的重要发病机制之一。
4、以β-catenin为中心的WNT/β-catenin信号通路参与了BPD的发生。
5、WNT/β-catenin信号通路参与BPD是通过激活其下游基因LEF-1实现的。
6、MF可能是肺发育及BPD中LF的主要存在形式。
7、WNT/β-catenin信号通路的活化可能与MF的增殖、活化有关。
8、WNT/β-catenin信号通路促进MF过度增殖活化、抑制其向肺泡间隔迁徙可能是其在BPD中的作用机制。
Introduction
Bronchopulmonary dysplasia(BPD) is a multifactorial disease resulting from the impact of injury(including oxygen toxicity,barotrauma,volutrauma,and infection) on the immature lung.It is one of the most common and significant medical complications associated with preterm birth.Advances in perinatal medicine have resulted in increasing numbers of very low birth weight preterm infants who are at risk of BPD, and the incidence of BPD has increased.The histopathologic changes of severe airway injury and alternating sites of overinflation and fibrosis which used to be seen in older forms of BPD have been replaced by a milder form characterized by alveolar hypoplasia and variable interstitial cellularity and/or fibroproliferation.
Mechanism of BPD included immature of the lung,injury and failured repairment after the injury.And we can comprehend it simply as the injury disturbed normal lung development and caused disorder of the repair.Injury caused by oxygen treatment and mechanical ventilation,oxygen toxicity and inflammation are thought to be the most contributing factors in the pathogenesis in BPD.But during the past years,treatments to reduce the injury such as antioxidant and anti-inflammatory have made no exact therapeutic effect.Further more,unlike injury to the adult lung that is essentially growth arrested,BPD indeed occurs in a growing lung with uncompleted morphogenesis.The spotlight focusing on impaired septation as a prominent feature of BPD prompts the neonatologist to question about disorders in the underlying molecular mechanisms,which are not only necessary for resolution and lung injury repair,but also for lung morphogenesis and development.Lung fibroblasts(LF) which can secrete elastin and participate in alveolarition,also can excrete collagen protein.and participate in lung fibrosis.It seems that LF plays an important role in the pathogenesis of BPD.
The Wnt pathway has been identified as one of the numerous signaling pathways critical for precise temporal and spatial control of lung morphogenesis.The centrality ofβ-catenin as a key regulatory protein in the Wnt cascade is conferred by its capacity to tightly regulate nuclear transcription.Conditional targeted deletion ofβ-catenin from the alveolar epithelium of developing mouse embryos results in complete disruption of peripheral terminal alveolar saccule formation and disturbances of pulmonary vasculogenesis.β-catenin activation has been implicated in several chronic pulmonary disorders,like idiopathic pulmonary fibrosis(IPF) et al.
We hypothesized thatβ-catenin signaling is involved in the reparative remodeling response to lung injury on an immature lung and actived in LE In this study,BPD was induced by hyperoxia exposure on neonatal rats which has been widely used for more than 20 years as a model to study associated cell and molecular alterations.We studied the expression of canonicalβ-catenin pathway molecules in BPD animal model and in fibroblasts from hyperoxia-exposed neonate rats.
Material and Methods
1.Animal models
Several litters of Wistar pups were pooled together within 12 hours after birth and randomly divided into two groups:group A is the hyperoxia-exposed group and group B is the air-exposed group.Rats in group A were placed in an oxygen chamber into which oxygen was continuously delivered(FiO2=0.85+0.02)(OM-25ME oxygen monitor,USA).CO2 were kept below 0.5%(Dapex Gas monitor,USA).Temperature and humidity were maintained at 22～25℃and 60～70%,respectively.The chamber was opened for＜15min daily to switch dams between air and O2 environment to avoid the dams from oxygen toxicity.Except the FiO2,other details of the method and experimental control factors were similar in these two groups.
2.Preparation of Lung Samples
Pups from each group were killed on days 3,7,and 14,and a tracheal cannula was placed.An abdominal incision was made,the diaphragm was punctured carefully to collapse the lungs.Left lung was inflation-fixed via tracheal cannula using 4% paraformaldehyde for morphology observation and immunohistochemistry study.In some cases,after midline thoracotomy,blood was collected from right ventricle for blood cell count,then the pulmonary artery was cannulated and the left atrial appendage was clipped and the lungs were gently perfused with 10 ml of 0.9%saline to remove blood.Then put lungs in RNase-free Eppendorf tubes and stored at-80℃freezer for RT-PCR and Western blotting.
3.Cell culture
Cultures of rat LF were established by enzymatic dissociation of finely minced lung tissue removed from pups of each group on days 3,7,14.Experiments were performed with fibroblasts in the second or third passages.
4.Experimental methods
(1)The appearance and weight were monitored everyday.
(2)Morphology observation:histological study,elastin staining,Masson staining.
(3)Immunohistochemistry:measurement the expression levels ofα-SMA andβ-catenin.
(4)Western blotting:measurement ofβ-catenin protein expression.
(5)RT-PCR:measurement ofβ-catenin,TCF-1,LEF-1 mRNA expression.
(6)Fibroblast was cultured and detected by morphology observation through inverted microscope and immunofluorescence staining to vimentin(a marker of LF). MTT method was used to evaluate the proliferative activity of LF.
(7)Immuocytochemistry:measurement the expression levels ofα-SMAβ-catenin and vimentin.
(8)Western blotting:measurement ofβ-catenin protein expression.
(9)RT-PCR:measurement ofβ-catenin,TCF-1,LEF-1 mRNA expression.
5.Statistical analysis
Normally distributed data are expressed as the mean±SEM and were assessed for significance by Student's t test or ANOVA with post-hoc continuity correction for multiple comparisons as indicated in the text.Non-normally distributed data were assessed for significance using the Wilcoxon rank sum test.Statistical calculations were performed using SPSS13.0 so,ware.Statistical difference was accepted at p＜0.05.
Results
1.General status and weight changes
Rats from group A presented dyspnea since 3 days exposure to hyperoxia.On the 7~(th) day of oxygen exposure they began to appear fatigue,pale,and present tachypnea and cyanosis for different degree.After 14 days exposure 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 on 3~(rd) day.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).
2.Lung morphology
(1)Changes of lung pathology.On the 3rd day of the experiment,the irregular structure of pulmonary alveoli with small alveolar lumen and thick alveolar septum was observed in each group.On the 7th day,alveoli structure of the control group became regular.While in the hyperxia group,the septum became thinner and decreased in their numbers,large numbers of inflammatory cells infiltrated into the alveoli and septum.On the 14th days,alveoli were regular in size in the control group.However in the hyperxia group,the alveolar cavity became larger,along with thinned alveolar septum,decreased numbers of alveoli,and increased numbers of local pulmonary interstitial fibroblasts.
(2)Changes of elastin expression.Elastin was expressed on the tips of septation between two alveolars,like mushrooms in group B on the 7th and 14th day.But expressed in the thick alveolar septa on the same experiment times.
(3)Masson staining.Lungs in group B had thin alveolar septa and minimal collagen staining.Lungs in group A had fine strands or thick bundles of collagen in the thick alveolar septa on day 14.
3.LF identification,proliferative activity
LF was determined by its morphology:fusiform shape,has long apophysis and orbicular-ovate nucleus on the centre;distributed like corona radiate or palisade. Vimentin staining was positive.
MTT method showd that LF from group A grown quicker than which from group B at eath experiment point.
4.Changes ofα-SMA protein expression
On lung tissue,α-SMA expressed on myofibroblast and vascular smooth muscle cell.In group B,it was expressed on the tips of septation between two alveolars on the 7th and 14th day,as elastin staining.But in group A,it was expressed in the thick alveolar septa on the same experiment times,as collagen staining.
On LF,it was expressed in the cytoplasm in almost ce11(＞95%),and increased in group A on day 7 and 14.
5.Changes ofβ-catenin expression
The expression was increased in total lung expression ofβ-catenin after 3 days' hyperoxia exposure.This peak occurred on the 14th day.Increased interstitial cell-predominant immunostaining ofβ-catenin was detected by light microscopic evaluation of immunostained lung sections on the 7th and 14th days in hyperoxia group compared with control.And it was observed that significantly increased nuclear colocalization ofβ-catenin in lung interstitial cells on the 7th and 14th days in hyperxia group compared with 21%02 control(P＜0.01).In the cultured LF,the expression ofβ-catenin was significantly increased on each time points after hyperxia exposure,and nuclear colocalization was obviously.
6.Changes of TCF-1 and LEF-1 mRNA expression
β-actin normalized mRNA expression by RT-PCR of LEF-1 was increased on the 3~(rd),7~(th) and 14t~(th) days in hyperxia group and LF from those groups,which were consistent with the period of maximalβ-catenin expression.While 13-actin normalized mRNA expression by RT-PCR of TCF-1 had no change during each experiment time point.
Conclusions
1.The histopathological changes of lung injury induced by hyperoxia(FIO_2=0.85) are similar to the characteristics of BPD in preterm infants.
2.Disturbed distribution of MF may connected with the alveolarition.
3.The proliferation of MF is connected with the repairment after lung injury,and thus made a very important role of MF in the development of BPD.
4.Wnt/β-catenin pathway is involved in alveolarition and the development of BPD.
5.Wnt/β-catenin pathway worked through 13-catenin/LEF-1.
6.LF worked as MF in the alveolarition and the development of BPD.
7.Wnt/β-catenin pathway may be involved in the proliferation and activation of MF.
8.Wnt/β-catenin worked in BPD maybe through the mechanism which involved in the proliferation,activation and migration of the MF.
支气管肺发育不良(Bronchopulmonary dysplasia,BPD)是采用机械通气或氧气治疗早产儿严重心肺疾病后常见的并发症,是威胁早产儿健康和生命质量的首要疾病。随着围产医学的不断发展,极低出生体重儿的成活率明显提高,BPD的发病率也在不断上升,其病理特点由过去显著的肺间质纤维化转变为以肺泡发育阻滞为突出特征。
BPD的发病机理包括:肺脏发育极不成熟,外界损伤以及损伤后修复机制的破坏。故此,BPD的发生可简单理解为在正常肺发育过程中,外界损伤导致的肺发育受阻和异常修复。以往的研究多着重于对肺组织损伤机制的研究,已证实氧毒性和机械通气介导的损伤、炎症反应在BPD的发生过程中具有重要作用,但抗炎、抗氧化的治疗对BPD却没有取得确切的疗效。随着对BPD认识的不断深入,对于即参与正常肺发育又调控肺组织损伤修复机制的研究,成为研究BPD发病机制的新视野。已证实肺发育过程中,位于肺泡分隔顶端的弹性蛋白不断向前延伸发展,是肺泡逐渐形成的物质基础;而细胞外基质的异常分布、沉积则是纤维化发生的直接原因。与两者的发生均密切相关的肺成纤维细胞(lung fibroblast,LF)的作用日益受到人们的关注。LF的增殖活化是其执行生物学功能的基础,对调节其增殖活化的信号通路的认识将有助于我们进一步理解LF的功能及其在BPD发生中的意义。
WNT信号通路是近年来才逐渐被人们认识到的一类控制细胞生长、增殖、传递细胞间相互调控信息的信号通路。其中WNT/β-catenin信号通路作为经典的WNT信号通路,已成为发育生物学及肿瘤学研究的热点。大量的研究证实,此信号通路活化的关键在于胞浆中是否存在结构稳定的β-catenin,以及其向胞核的转位。已有研究表明,早期肺发育(即支气管树形成)过程中WNT/β-catenin信号通路发挥了重要作用;且在对成人肺纤维化疾病的研究中发现WNT/β-catenin信号通路可能起着关键性的作用。但在肺泡化过程中WNT/β-catenin信号通路是否存在,其作用如何,在肺泡化障碍及肺纤维化的早产儿BPD中有何作用?到目前为止,鲜有报道。既然WNT/β-catenin信号通路参与调节肺发育及肺纤维化,且与细胞增殖分化有关,那么其在BPD中的作用是否通过促进LF增殖活化来实现?
本研究拟从组织和细胞水平,观察WNT/β-catenin信号通路关键因子β-catenin及其下游基因TCF-1/LEF-1的活化情况,探讨WNT/β-catenin信号通路在BPD中的作用及其机制,完善BPD的发病机制,为BPD的预防和治疗提供一个新思路。
实验材料与方法
一、动物模型
新生Wistar大鼠生后12h之内随机分为高氧组(A组)和空气组(B组)。A组置于玻璃氧箱中,持续输入氧气,FiO_2=0.85±0.02(美国OM-25ME型测氧仪监测)。用钠石灰吸收CO_2,使其浓度＜0.5%(Dapex气体分析仪),温度22～25℃,湿度60%～70%。每天定时开箱15min,添水、饲料及更换垫料,并与空气组交换母鼠以免因氧中毒而致喂养能力下降。B组置于空气中(FiO_2=0.21),余控制因素同A组。
二、标本的采集和处理
每组分别于实验后3、7、14d随机选取8只称重,5%哥拉腹腔注射(0.6ml/100mg)麻醉后,分离气管,打开胸腔,暴露心肺,经气管缓慢注入4%多聚甲醛,然后置于4%多聚甲醛中,4℃过夜,石蜡包埋,制作4μm组织切片,用于肺形态学观察、免疫组织化学检测。或于打开胸腔后,剪开左心耳,经右心室插入套管至肺动脉,注入10ml生理盐水洗净肺内残血,收集肺组织,置于无RNase的Eppendorf管中,液氮速冻,—80℃冰箱保存,用于RT-PCR及Western-blot检测。
三、细胞培养
动物模型建立后,分别于实验第3、7、14天取每组2-3只动物进行肺组织LF原代培养,取传2-3代细胞进行检测。
四、实验方法
1、组织水平
(1)动态观察各组新生大鼠的一般状态、监测体重。
(2)肺形态学观察:①H&E染色;②弹性蛋白染色;③Masson染色。
(3)免疫组织化学技术:α-平滑肌动蛋白(α-smooth muscle actin,α-SMA)、β-catenin蛋白检测。
(4)Western-blot法:检测肺组织β-catenin蛋白表达。
(5)RT-PCR法:检测肺组织α-表达。
2、细胞水平
(1)细胞培养及鉴定,MTT法检测细胞增殖情况。
(2)免疫细胞化学技术:α-平滑肌动蛋白(α-smooth muscle actin,α-SMA)、β-catenin、波形蛋白检测。
(3)免疫荧光:波形蛋白染色进行细胞鉴定。
(4)Western-blot法:检测细胞β-catenin蛋白表达。
(5)RT-PCR法:检测肺LF中β-catenin、TCF-1、LEF-1表达。
五、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((?)+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 ftest。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
A组新生鼠3d时肤色尚红润,脱离氧气后呼吸稍促,7d时出现少动、皮肤苍白以及离氧后呼吸频率稍快伴不同程度的发绀,随高氧暴露时间延长而逐渐加重,在14d时反应差,不活泼,体毛涩、无光泽,时有头颤;离氧后呼吸增快、出现呼吸困难、头颤明显,口周发绀。恢复氧供后可缓解。而B组始终反应较好,肤色红润,体重平稳增长,14天时睁眼。
A组在高氧暴露7d开始,体重与B组相比下降(p＜0.05),随着高氧暴露时间的延长,体重差异更加显著(p＜0.01)。
二、肺形态学改变
1、肺组织病理变化
A组3d肺泡结构紊乱,间隔少量炎性细胞浸润;7d间隔有大量炎细胞浸润,肺泡腔有渗出,肺灶状出血;14d肺泡数量明显减少,形成肺大泡,间隔增厚,肺间质胶原沉积。B组14d肺泡大小均匀一致,肺泡间隔较薄。
2、弹性蛋白染色
结果显示B组7天、14天时弹性蛋白主要表达于次级隔顶端,呈“蘑菇”样改变。A组则主要表达于肺间质。
3、Masson染色
结果显示B组肺泡间隔较薄,少量蓝色胶原沉积,A组14d蓝色胶原呈条索状或呈束状沉积于增厚的肺间质内。
三、细胞鉴定及细胞增殖测定
按Kelleher等建立的方法分离、培养LF,并进行鉴定:1、形态学鉴定:倒置显微镜下LF呈梭形,有较长的突起,胞核呈卵圆形,位于细胞中央,呈放射状或栅栏状排列;2、免疫荧光:波形蛋白表达于胞浆,绿色荧光为波形蛋白阳性染色;3、免疫细胞化学染色:胞浆内棕黄色颗粒为阳性染色。
MTT结果显示:A组LF生长较B组明显增加,实验各时间点均具有显著差异(p＜0.01)。
四、α-SMA蛋白在肺组织及LF中表达的动态变化
α-SMA主要表达于MF及血管平滑肌细胞。B组α-SMA 7天、14天时主要表达于次级隔顶端,与弹性蛋白表达部位基本一致,间质少有表达。A组7dα-SMA表达开始增加(p＜0.01),表达部位主要在间质,14d在上述部位表达明显增强(p＜0.01),与胶原蛋白表达一致性。在培养的LF中,胞浆中均存在α-SMA的阳性表达(＞95%),表达强度随高氧暴露时间延长而增强,7d、14d与B组存在显著性差异(p＜0.01)。
五、β-catenin在肺组织及LF中的表达
1、β-catenin蛋白在肺组织中的动态表达
免疫组化结果显示:B组3天肺组织支气管上皮细胞、部分肺泡上皮细胞、间质细胞胞核内均出现β-catenin表达,随着生后日龄的增加,在次级隔表达明显增多。A组自吸氧3d开始在上述细胞胞核中出现高表达,与B组具有显著性差异(p＜0.05),7d、14d仍呈高表达(p＜0.01),间质细胞核中表达明显增多。
Western-blot结果与免疫组化结果基本一致,两组β-catenin表达随日龄增长均逐渐增加,A组增加更加显著。实验第3天始,两组β-catenin表达就出现显著性差异(p＜0.05),7天及14天差异更为显著(p＜0.01)。
2、β-catenin基因在肺组织中的动态表达
RT-PCR结果显示:两组β-catenin基因表达随日龄增长逐渐增加,A组基因表达显著,其变化规律与蛋白表达趋势基本一致。两组内各时间点差异显著(p＜0.05)。
3、β-catenin蛋白在LF中的动态表达
免疫细胞化学结果显示:两组β-catenin均表达于细胞核内。
Western-blot结果显示:两组细胞β-catenin表达随日龄增长逐渐增加,A组增加更加显著,其变化规律与组织水平表达趋势基本一致。
4、β-catenin基因在LF中的动态表达
RT-PCR结果显示:两组β-catenin基因表达随日龄增长逐渐增加,A组基因表达增长显著,其变化规律与蛋白表达趋势基本一致。两组内各时间点差异显著(p＜0.05)。
六、TCF-1、LEF-1 mRNA表达的变化
1、TCF-1/LEF-1 mRNA在肺组织中的表达变化
RT-PCR结果显示:LEF-1 mRNA在A组自吸氧3d开始表达明显增强(p＜0.01),7d、14d仍呈高表达,与B组有明显差异(p＜0.05)。两组TCF-1 mRNA表达无明显差异(p＞0.05)。
2、TCF-1/LEF-1 mRNA在LF中的表达变化
RT-PCR结果显示:两组细胞中TCF-1/LEF-1 mRNA表达与组织水平变化趋势一致。随日龄增长逐渐增加,LEF-1 mRNA在A组3d表达增强(p＜0.01),7d、14d仍呈高表达,两组表达具有明显差异(p＜0.05),而两组TCF-1 mRNA在实验各时点表达无明显差异(p＞0.05)。
结论
1、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BPD的发生发展特点和病理学改变。
2、肺肌成纤维细胞的异常分布(未到达肺泡分隔顶端而沉积于间质)可能是BPD肺泡发育障碍的机制之一。
3、肺肌成纤维细胞沉积于间质并过度表达,与BPD肺泡损伤后修复障碍密切相关。肺肌成纤维细胞在BPD中的异常分布、表达可能是BPD的重要发病机制之一。
4、以β-catenin为中心的WNT/β-catenin信号通路参与了BPD的发生。
5、WNT/β-catenin信号通路参与BPD是通过激活其下游基因LEF-1实现的。
6、MF可能是肺发育及BPD中LF的主要存在形式。
7、WNT/β-catenin信号通路的活化可能与MF的增殖、活化有关。
8、WNT/β-catenin信号通路促进MF过度增殖活化、抑制其向肺泡间隔迁徙可能是其在BPD中的作用机制。
Introduction
Bronchopulmonary dysplasia(BPD) is a multifactorial disease resulting from the impact of injury(including oxygen toxicity,barotrauma,volutrauma,and infection) on the immature lung.It is one of the most common and significant medical complications associated with preterm birth.Advances in perinatal medicine have resulted in increasing numbers of very low birth weight preterm infants who are at risk of BPD, and the incidence of BPD has increased.The histopathologic changes of severe airway injury and alternating sites of overinflation and fibrosis which used to be seen in older forms of BPD have been replaced by a milder form characterized by alveolar hypoplasia and variable interstitial cellularity and/or fibroproliferation.
Mechanism of BPD included immature of the lung,injury and failured repairment after the injury.And we can comprehend it simply as the injury disturbed normal lung development and caused disorder of the repair.Injury caused by oxygen treatment and mechanical ventilation,oxygen toxicity and inflammation are thought to be the most contributing factors in the pathogenesis in BPD.But during the past years,treatments to reduce the injury such as antioxidant and anti-inflammatory have made no exact therapeutic effect.Further more,unlike injury to the adult lung that is essentially growth arrested,BPD indeed occurs in a growing lung with uncompleted morphogenesis.The spotlight focusing on impaired septation as a prominent feature of BPD prompts the neonatologist to question about disorders in the underlying molecular mechanisms,which are not only necessary for resolution and lung injury repair,but also for lung morphogenesis and development.Lung fibroblasts(LF) which can secrete elastin and participate in alveolarition,also can excrete collagen protein.and participate in lung fibrosis.It seems that LF plays an important role in the pathogenesis of BPD.
The Wnt pathway has been identified as one of the numerous signaling pathways critical for precise temporal and spatial control of lung morphogenesis.The centrality ofβ-catenin as a key regulatory protein in the Wnt cascade is conferred by its capacity to tightly regulate nuclear transcription.Conditional targeted deletion ofβ-catenin from the alveolar epithelium of developing mouse embryos results in complete disruption of peripheral terminal alveolar saccule formation and disturbances of pulmonary vasculogenesis.β-catenin activation has been implicated in several chronic pulmonary disorders,like idiopathic pulmonary fibrosis(IPF) et al.
We hypothesized thatβ-catenin signaling is involved in the reparative remodeling response to lung injury on an immature lung and actived in LE In this study,BPD was induced by hyperoxia exposure on neonatal rats which has been widely used for more than 20 years as a model to study associated cell and molecular alterations.We studied the expression of canonicalβ-catenin pathway molecules in BPD animal model and in fibroblasts from hyperoxia-exposed neonate rats.
Material and Methods
1.Animal models
Several litters of Wistar pups were pooled together within 12 hours after birth and randomly divided into two groups:group A is the hyperoxia-exposed group and group B is the air-exposed group.Rats in group A were placed in an oxygen chamber into which oxygen was continuously delivered(FiO2=0.85+0.02)(OM-25ME oxygen monitor,USA).CO2 were kept below 0.5%(Dapex Gas monitor,USA).Temperature and humidity were maintained at 22～25℃and 60～70%,respectively.The chamber was opened for＜15min daily to switch dams between air and O2 environment to avoid the dams from oxygen toxicity.Except the FiO2,other details of the method and experimental control factors were similar in these two groups.
2.Preparation of Lung Samples
Pups from each group were killed on days 3,7,and 14,and a tracheal cannula was placed.An abdominal incision was made,the diaphragm was punctured carefully to collapse the lungs.Left lung was inflation-fixed via tracheal cannula using 4% paraformaldehyde for morphology observation and immunohistochemistry study.In some cases,after midline thoracotomy,blood was collected from right ventricle for blood cell count,then the pulmonary artery was cannulated and the left atrial appendage was clipped and the lungs were gently perfused with 10 ml of 0.9%saline to remove blood.Then put lungs in RNase-free Eppendorf tubes and stored at-80℃freezer for RT-PCR and Western blotting.
3.Cell culture
Cultures of rat LF were established by enzymatic dissociation of finely minced lung tissue removed from pups of each group on days 3,7,14.Experiments were performed with fibroblasts in the second or third passages.
4.Experimental methods
(1)The appearance and weight were monitored everyday.
(2)Morphology observation:histological study,elastin staining,Masson staining.
(3)Immunohistochemistry:measurement the expression levels ofα-SMA andβ-catenin.
(4)Western blotting:measurement ofβ-catenin protein expression.
(5)RT-PCR:measurement ofβ-catenin,TCF-1,LEF-1 mRNA expression.
(6)Fibroblast was cultured and detected by morphology observation through inverted microscope and immunofluorescence staining to vimentin(a marker of LF). MTT method was used to evaluate the proliferative activity of LF.
(7)Immuocytochemistry:measurement the expression levels ofα-SMAβ-catenin and vimentin.
(8)Western blotting:measurement ofβ-catenin protein expression.
(9)RT-PCR:measurement ofβ-catenin,TCF-1,LEF-1 mRNA expression.
5.Statistical analysis
Normally distributed data are expressed as the mean±SEM and were assessed for significance by Student's t test or ANOVA with post-hoc continuity correction for multiple comparisons as indicated in the text.Non-normally distributed data were assessed for significance using the Wilcoxon rank sum test.Statistical calculations were performed using SPSS13.0 so,ware.Statistical difference was accepted at p＜0.05.
Results
1.General status and weight changes
Rats from group A presented dyspnea since 3 days exposure to hyperoxia.On the 7~(th) day of oxygen exposure they began to appear fatigue,pale,and present tachypnea and cyanosis for different degree.After 14 days exposure 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 on 3~(rd) day.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).
2.Lung morphology
(1)Changes of lung pathology.On the 3rd day of the experiment,the irregular structure of pulmonary alveoli with small alveolar lumen and thick alveolar septum was observed in each group.On the 7th day,alveoli structure of the control group became regular.While in the hyperxia group,the septum became thinner and decreased in their numbers,large numbers of inflammatory cells infiltrated into the alveoli and septum.On the 14th days,alveoli were regular in size in the control group.However in the hyperxia group,the alveolar cavity became larger,along with thinned alveolar septum,decreased numbers of alveoli,and increased numbers of local pulmonary interstitial fibroblasts.
(2)Changes of elastin expression.Elastin was expressed on the tips of septation between two alveolars,like mushrooms in group B on the 7th and 14th day.But expressed in the thick alveolar septa on the same experiment times.
(3)Masson staining.Lungs in group B had thin alveolar septa and minimal collagen staining.Lungs in group A had fine strands or thick bundles of collagen in the thick alveolar septa on day 14.
3.LF identification,proliferative activity
LF was determined by its morphology:fusiform shape,has long apophysis and orbicular-ovate nucleus on the centre;distributed like corona radiate or palisade. Vimentin staining was positive.
MTT method showd that LF from group A grown quicker than which from group B at eath experiment point.
4.Changes ofα-SMA protein expression
On lung tissue,α-SMA expressed on myofibroblast and vascular smooth muscle cell.In group B,it was expressed on the tips of septation between two alveolars on the 7th and 14th day,as elastin staining.But in group A,it was expressed in the thick alveolar septa on the same experiment times,as collagen staining.
On LF,it was expressed in the cytoplasm in almost ce11(＞95%),and increased in group A on day 7 and 14.
5.Changes ofβ-catenin expression
The expression was increased in total lung expression ofβ-catenin after 3 days' hyperoxia exposure.This peak occurred on the 14th day.Increased interstitial cell-predominant immunostaining ofβ-catenin was detected by light microscopic evaluation of immunostained lung sections on the 7th and 14th days in hyperoxia group compared with control.And it was observed that significantly increased nuclear colocalization ofβ-catenin in lung interstitial cells on the 7th and 14th days in hyperxia group compared with 21%02 control(P＜0.01).In the cultured LF,the expression ofβ-catenin was significantly increased on each time points after hyperxia exposure,and nuclear colocalization was obviously.
6.Changes of TCF-1 and LEF-1 mRNA expression
β-actin normalized mRNA expression by RT-PCR of LEF-1 was increased on the 3~(rd),7~(th) and 14t~(th) days in hyperxia group and LF from those groups,which were consistent with the period of maximalβ-catenin expression.While 13-actin normalized mRNA expression by RT-PCR of TCF-1 had no change during each experiment time point.
Conclusions
1.The histopathological changes of lung injury induced by hyperoxia(FIO_2=0.85) are similar to the characteristics of BPD in preterm infants.
2.Disturbed distribution of MF may connected with the alveolarition.
3.The proliferation of MF is connected with the repairment after lung injury,and thus made a very important role of MF in the development of BPD.
4.Wnt/β-catenin pathway is involved in alveolarition and the development of BPD.
5.Wnt/β-catenin pathway worked through 13-catenin/LEF-1.
6.LF worked as MF in the alveolarition and the development of BPD.
7.Wnt/β-catenin pathway may be involved in the proliferation and activation of MF.
8.Wnt/β-catenin worked in BPD maybe through the mechanism which involved in the proliferation,activation and migration of the MF.
引文
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