胰岛素样生长因子-Ⅰ在Nitrofen诱导的先天性膈疝大鼠模型胎肺中的表达

目的 研究IGF-Ⅰ在先天性膈疝胎肺上的表达及探讨其在CDH肺发育不良中的作用。方法 采用免疫组化和图像分析方法检测IGF-Ⅰ在胎肺中的表达及相对含量。结果IGF-Ⅰ在对照组表达微弱或缺如，在实验组明显表达于细支气管上皮细胞的胞浆及部分神经丛，其相对含量显著高于对照组。IGF-Ⅰ与平均肺泡面积具有显著的负相关。结论 CDH肺发育不良可能是原发性；IGF-Ⅰ的表达增高，具有组织特异性；IGF-Ⅰ与肺泡面积呈负的直线相关性。     

先天性膈疝肺动脉高压的实验研究

目的研究先天性膈疝（Congenital diaphragmatic hernia,CDH）肺发育不良中肺动脉高压（Dulmonary hypertension,PH）的量化指标,探讨血管内皮生长因子（vascular endothelial growth factor,VEGF）在CDH胎肺中的表达及在CDH肺动脉高压发生中的可能作用,探讨地塞米松（Dexamethasone,Dex）对VEGF和CDH肺发育的影响。方法采用Nitrofen诱导CDH模型胎鼠并分组：Dex组予以产前Dex治疗,CDH组未给予Dex,另取正常胎鼠为对照组。采用组织学测量、肺蛋白含量检测等方法,测定出平均肺泡面积、平均肺泡间隔厚度、每高倍视野肺血管数、动脉中膜厚度占外径比（MT％）、管壁面积占血管总面积比（WA％）、管腔面积占血管总面积比（LA％）、左肺与体重比、左肺蛋白含量与体重比等;实时荧光定量PCR（QPCR）和免疫蛋白印迹（Western blotting）方法检测各组胎肺中VEGF的表达及相对含量。结果与对照组相比,CDH组胎肺显著发育不良,包括肺微小动脉结构明显异常,表现为每高倍视野肺血管数减少、MT％增大、WA％增大,LA％减小（均P〈0．05）;另外VEGFmRNA及蛋白水平均明显升高（P〈0．05）。与CDH组相比,Dex组胎肺发育明显改善,表现为每高倍视野肺血管数增加、动脉中膜厚度占外径比减小、管腔面积占血管总面积比增大（均P〈0．05）;但VEGF mRNA及蛋白水平未发现明显变化（P〉0．05）。结论VEGF在CDH胎肺中的表达增高可能是CDH肺发育不良形成机制之一;产前地塞米松治疗可明显改善肺微小动脉结构异常及肺发育不良,但未发现对VEGF的表达有影响,提示Dex可能并非通过调节VEGF的表达而发挥改善肺动脉高压及肺发育不良的作用。     

Wnt7b在Nitrofen诱导的先天性膈疝大鼠模型胎肺中的表达

目的 研究Wnt7b在Nitrofen诱导的先天性膈疝(congenital diaphragmatic hernia,CDH)胎肺中的表达特点,探讨其在CDH肺发育不良发生机制中的可能作用.方法 采用实时荧光定量PCR方法检测Wnt7b基因在妊娠E17.5、E19.5、E21.5的Nitrofen诱导CDH大鼠模型胎肺及正常对照大鼠胎肺中的相对表达量,并用免疫组化方法检测Wnt7b蛋白在胎肺中的表达.结果 正常对照组和CDH组胎肺中Wnt7b mRNA的表达水平均随着胎龄的增加呈下降趋势,其中E17.5胎龄CDH组胎肺中Wnt7b mRNA的表达水平与对照组相比,差异无统计学意义;而E19.5、E21.5胎龄CDH组胎肺中Wnt7b mRNA的表达水平高于对照组,差异有统计学意义(P＜0.05).对E21.5胎龄对照组和CDH组的标本进行免疫组化检测,结果显示Wnt7b免疫阳性细胞主要分布在支气管和细支气管上皮,且CDH组Wnt7b免疫阳性程度(以平均光密度值表示)较正常对照组升高,有统计学差异.结论 Wnt7b mRNA表达量随大鼠胎肺逐渐发育成熟而下降,提示其在胎肺中的表达量与胎肺发育的成熟度有关.CDH组中E19.5、E21.5胎龄时Wnt7b mRNA的表达水平明显高于对照组,是CDH孕晚期胎肺发育滞后的分子基础之一,提示Wnt7b在CDH孕晚期胎肺中表达增高可能参与了肺发育不良.Wnt7b蛋白在肺发育中特异性定位于气道上皮,提示信号町能在肺上皮与间质细胞之间有重要联系,协同促进上皮和间质的发育.     

缺氧诱导丝裂原因子在先天性膈疝胎鼠肺中的表达

目的研究缺氧诱导丝裂原因子（hypoxia—inducedmitogenicfactor，HIMF）在先天性膈疝（congenitaldiaphragmatichernia，CDH）胎鼠肺组织中的表达，探讨其在CDH肺发育不良中的作用。方法实验组10只BABL／C小鼠妊娠8d时经胃管注入25mg除草醚，正常对照组给予食用油，妊娠21d行剖腹产，解剖胎鼠两侧肺组织，采用免疫组化、Westernblot方法检测HIMF表达。结果实验组CDH致畸率56．5％，肺发育不良，处于假腺体期和原始肺小管期，HIMF蛋白显著表达下调（P〈0．05）；实验组内产生CDH者胎肺内HIMF表达水平与无CDH者比较差异无显著性意义（P〉0．05）；CDH膈疝侧与非膈疝侧肺组织HIMF表达差异无显著性意义（P〉0．05）。结论在CDH肺发育不良组织中HIMF蛋白表达显著下调，且早于膈疝形成，可能参与CDH肺发育不良的发病机制。     

Hoxa5基因在先天性膈疝大鼠模型胎肺中的表达

目的 研究Hoxa5基因在先天性膈疝(congenital diaphragmatic hernia,CDH)胎肺中的表达特点以及探讨其在CDH肺发育不良发生机制中的可能作用.方法 采用实时荧光定量PCR(real time quantitative PCR,QPCR)方法检测Hoxa5基因在妊娠第17.5天、19.5天、21.5天的nitrofen诱导CDH大鼠模型胎肺及正常对照大鼠胎肺巾的相对表达量.结果 正常对照组胎肺中Hoxa5 mRNA的表达水平随着胎龄的增加旱下降趋势,其中第21.5天胎肺中Hoxa5 mRNA的表达水平显著下降,与其他二胎龄点相比差异有统计学意义(P＜0.05);CDH组中Hoxa5 mRNA的表达趋势与正常胎肺相似,即随胎龄的增加其表达量下降,其当中第21.5天胎肺中Hoxa5 mRNA的表达水平明显降低,与其他二胎龄点相比差异有统计学意义(P＜0.05).第17.5天与19.5天胎龄CDH组胎肺中Hoxa5 mRNA的表达水平分别与相应对照组相比,差异无统计学意义;而妊娠晚期(第21.5天胎龄)CDH组胎肺中Hoxa5 mRNA的表达水平高于对照组.差异具有统计学意义.结论 正常对照组中第21.5天胎龄时Hoxa5 mRNA的表达水平显著降低,提示Hoxa5 mRNA在孕晚期的低表达是正常肺发育的分子基础之一;而CDH组中第21.5天胎龄时Hoxa5 mRNA的表达水平明显高于对照组,提示Hoxa5在孕晚期CDH胎肺中Hoxa5的高表达可能是CDH肺发育不良形成机制之一.     

汉防己甲素对先天性膈疝大鼠的保护作用

目的：探讨汉防己甲素（TET）产前干预对先天性膈疝（CDH）大鼠的保护作用及机制。方法：将妊娠Sprague-Dawley大鼠随机分为对照组、除草醚组与TET治疗组。后两组孕 9.5 d 时采用除草醚灌胃法建立 CDH 大鼠模型;治疗组自孕18.5 d 起给予 TET 灌胃（每日30 mg/kg,连续3 d）;21 d 对部分孕鼠行剖腹并抽取羊水,观察胎鼠膈疝形成情况。采用 ELISA 法、免疫组化染色法检测羊水和胎肺中 TNF-α 的表达情况。余孕鼠自然分娩,观察各组仔鼠出生后情况。结果：除草醚组胎鼠无论有无膈疝形成均存在肺发育不良,肺及羊水中 TNF-α 的表达均明显升高;TET治疗组胎鼠巨大膈疝的发生率低于除草醚组,肺与羊水中 TNF-α的含量明显较除草醚组少（P＜0.01）。在自然分娩的仔鼠中,TET治疗组仔鼠的 24 h存活率明显高于除草醚组（P＜0.01）。结论：产前应用 TET 能降低CDH大鼠模型胎肺与羊水中 TNF-α 的含量,改善因除草醚诱导的胎鼠肺发育不良,减少巨大膈疝的发生,提高仔鼠的存活率。     

Nitrofen对膜受体STRA6在先天性膈疝大鼠模型中表达的影响

目的 研究STRA6基因在先天性膈疝胎肺中的表达特点并探讨其在CDH肺发育不良发生机制中的可能作用.方法 采用实时荧光定量PCR(real time quantitative PCR,QPCR)方法检测在妊娠D 15.5、D 17.5、D21.5的Nitrofen诱导CDH大鼠模型胎肺及正常对照组大鼠胎肺中的STRA6基因mRNA相对表达量.结果 control组:STRA6 mRNA相对表达量呈下降趋势,各时期的相对表达量分别为14.800±5.155,4.061±2.765,1.000±0.449,D 15.5与D 17.5和D 21.5相比有统计学意义(P=0.000＜0.01);Nitrofen组:STRA6 mRNA相对表达量波动.各时期的相对表达量分别为1.191±0.351、7.552±3.716、0.951±0.942、D17.5表达升高,D17.5与D15.5和D21.5相比差异有统计学意义(P=0.000＜0.01);在D 15.5,controls组与Nitrofen诱导组相比有统计学意义(P=0.000＜0.01).结论 在实验动物模型胎鼠肺发育早期(D15.5),Nitrofen干扰肺细胞表面RBP受体STRA6的表达.可能引起肺细胞吸收Vit A障碍而导致CDH肺发育不良的发生;在实验动物模型胎鼠肺发育中期(D 17.5),STRA6的表达增高,这可能是维A酸信号途径对肺细胞内的VitA水平低下的一种负反馈反应.     

SB265610抗炎症干预在新生大鼠高氧肺损伤中的抗氧化作用

目的：探讨受体拮抗剂SB265610干预中性粒细胞（PMN）趋化作用后，高氧暴露新生大鼠肺部自由基产生情况。方法：采用600mL/LO2建立新生大鼠高氧暴露支气管肺发育不良模型（BPD）；新生大鼠随机分为4组：空气对照组，空气＋SB265610组，高氧对照组，高氧＋SB265610组；通过免疫组织化学和支气管肺泡灌洗液（BALF）细胞计数检测肺部中性粒细胞趋化因子、细胞因子诱导的中性粒细胞趋化因子-1（CINC-1）表达和炎性细胞聚集的情况；应用气相色谱-质谱（CC-MS）联用技术测定肺组织羟自由基；酶联免疫法测定脂质过氧化产物8-异前列腺烷。结果：高氧对照组新生大鼠肺内巨噬细胞、支气管和肺泡上皮细胞中CINC-1表达水平升高，同时WBC和PMN计数较空气对照组显著增加（Pa〈0．01）；高氧对照组新生大鼠肺中羟自由基水平显著高于空气对照组（P〈0．01），SB265610处理后高氧暴露鼠肺组织羟自由基水平明显下降（P〈0、01）；高氧对照组新生大鼠肺组织8-异前列腺烷水平显著升高，SB265610处理后明显降低（P〈0．01）。结论：抗PMN趋化干预可明显降低新生大鼠高氧肺损伤过程中肺内羟自由基及脂质过氧化水平。     

汉防己甲素对于膈疝模型大鼠胎肺中血管内皮生长因子表达的影响北大核心CSCD

目的研究血管内皮生长因子（vascularendothelialgrowthfactor,VEGF）在大鼠膈疝模型胎肺中的表达特点及规律,以及汉防己甲素（Tet）干预后的变化。方法20只健康怀孕的SD大鼠雌鼠在孕9．5d时用随机数字表法随机分为3组：即正常对照组（C组）6只;膈疝组（D组）7只;膈疝汉防己甲素组（DT组）7只。D组和DT组灌胃给予除草醚（Nitrofen125mg／只,溶于2ml橄榄油中）,C组接受等量的橄榄油,DT组于孕11--13d每天给予30mg·kg^-1·d^-1Tet灌胃,C组和D组仅给予生理盐水。分别于第16d、18d和21d麻醉下剖宫取胎鼠双肺,应用组织学观察及原位杂交方法研究VEGF在膈疝模型不同时段胎鼠肺的表达特点及规律,及应用汉防己甲素后VEGF在膈疝模型不同时段胎鼠肺的表达特点及变化规律。结果本组实验中D组18d（D18）和21d（D21）的膈疝发生率为85．2％和DT组18d（DT18）和21d（DT21）的膈疝发生率为76．7％,差异无统计学意义（P〉0．05）。C组16d（C16）、18d（C18）和21d胎龄胎鼠（C21）胎肺VEGFmRNA分光光密度（integralopticaldensity,IOD）分别为7493．4±3167．9、4024．7±2204．9和8697．4±1466．8。D组16d（D16）、18d（D18）和21d胎龄胎鼠（D21）ga肺VEGFmRNAIOD分别为15269．2±5307．5、5670．5±1588．5和8061．3±2245．7。DT组16d（DT16）、18d（DT18）和21d胎龄胎鼠（DT21）胎肺VEGFmRNAI（）D分别为10742．8±4803．5、5626．4±3231．3和11687．7±11628．7。D18与D16和D21与D16的IOD比较差异有统计学意义（P〈0．05）。VEGFmRNA阳性表达的分布特点：C16、D16与DT16的胎肺VEGFmRNA阳性表达位于呼吸道内皮细胞胞浆,尤以远端呼吸道出芽区域更为显著。C18、D18、DT18、C21、D21与DT21的胎肺支气管壁黏膜和血管壁内皮细胞、血管壁肌层可见VEGFmRNA的阳性表达。C21胎肺VEGFmRNA阳性表达部位主要位于肺泡壁和肺问质,呈网状,而D21胎肺肺泡壁和肺间质处VEGFmRNA阳性表达不明显。DT21胎肺VEGFmRNA阳性表达部位主要位于肺泡壁和肺间质,呈网状与C21近似。结论①Nitrofen可能通过抑制VEGF的表达阻碍CDH模型大鼠胎肺晚期肺泡及其血管的发育。②应用汉防己甲素对CDH大鼠进行产前干预后,可使其实验胎鼠肺内VEGFmRNA的阳性分布恢复正常。提示汉防己甲素可能通过调节VEGF的表达而改善肺的发育。③汉防己甲素可能不能阻止Nitrofen诱导大鼠膈疝形成。     

脂肪型脂肪酸结合蛋白在早产大鼠高氧肺损伤时的表达

目的 观察脂肪型脂肪酸结合蛋白4(FABP4)在早产大鼠高氧肺损伤时肺组织及支气管肺泡灌洗液(BALF)中的表达,探讨其与新型支气管肺发育不良(BPD)发病机制之间的关系.方法 早产大鼠生后6 h 内随机分为高氧组和对照组,对照组置于常压空气中,高氧组置于浓度为60% 的高氧舱中,两组均于出生后第3 天(P3)、第7 天(P7)和第14 天(P14)各随机取8 只大鼠,采用免疫组织化学方法和逆转录-聚合酶链反应技术检测不同时间两组肺组织FABP4 蛋白及mRNA 表达水平,应用ELISA 方法检测BALF中FABP4 的含量.结果 FABP4 主要在肺泡巨噬细胞、支气管上皮细胞和血管内皮细胞表达.两组FABP4 蛋白和mRNA 在肺组织中的表达以及两组BALF 中FABP4 的含量均随鼠龄递增呈逐渐增加的趋势,至P14 时最高.高氧组肺组织中FABP4 mRNA 的表达在P7、P14,FABP4 蛋白的表达在P3、P7 及P14 时均高于对照组(均P<0.05);高氧组BALF 中FABP4 的含量在P7、P14 时均高于对照组(均P<0.05).结论 高氧肺损伤时FABP4 表达升高,可能是引起肺微血管发育障碍及肺泡化进程受阻,进而导致新型BPD 发生的重要因素.     

Disturbance of parathyroid hormone-related protein signaling in the nitrofen-induced hypoplastic lung

Purpose

Despite remarkable progress in resuscitation and intensive care, the morbidity and mortality rates in congenital diaphragmatic hernia (CDH) remain high due to severe pulmonary hypoplasia. The pathogenesis of pulmonary hypoplasia associated with CDH is still not clearly understood. Pulmonary parathyroid hormone-related protein (PTHrP) is expressed in the type II epithelial cells and stimulates surfactant production by a paracrine feedback loop regulated by PTHrP receptor (PTHrP-R), which is expressed in the mesenchyme, during terminal airway differentiation. It has been reported that PTHrP knockout and PTHrP-R null mice both exhibit pulmonary hypoplasia, disrupting alveolar maturation before birth. We designed this study to test the hypothesis that gene expression of PTHrP and PTHrP-R is downregulated in the late stages of lung morphogenesis in the nitrofen-induced hypoplastic lung.

Methods

Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetal lungs were harvested on D15, 18, and 21 and divided into three groups: control, nitrofen without CDH [CDH(?)], and nitrofen with CDH [CDH(+)] (n = 8 at each time point for each group, respectively). Total mRNA was extracted from fetal lungs and mRNA expression of PTHrP and PTHrP-R was analyzed by real-time RT-PCR and the significant differences between the groups were accepted at P < 0.05 by statistical analysis. Immunohistochemical studies were also performed to evaluate PTHrP and PTHrP-R protein expression at each time point.

Results

Pulmonary mRNA expression of PTHrP-R was significantly decreased in both nitrofen groups [CDH(?) and CDH(+)] compared to controls at D18 and 21. The mRNA level of PTHrP was significantly decreased at D21 in both nitrofen groups compared to controls. Immunoreactivity of PTHrP and PTHrP-R at D18 and 21 was diminished in the distal epithelium and in the mesenchyme, respectively, in the nitrofen-induced hypoplastic lung compared to control lungs. There were no significant differences in both gene/protein expression of PTHrP and PTHrP-R on D15.

Conclusion

Downregulation of PTHrP and PTHrP-R gene expression during late lung morphogenesis may cause pulmonary hypoplasia in the nitrofen CDH model, disrupting alveolar maturation and surfactant production by interfering with mesenchymal–epithelial interactions.     

大鼠胎肺内表皮生长因子及其受体变化与先天性膈疝肺发育不良的关系

目的观察表皮生长因子(EGF)及其受体(EGFR)在Nitrofen诱导的CDH模型胎肺中表达的情况,探讨CDH时肺发育不良的原因。方法实验组20只SD怀孕大鼠于孕9.5d时经胃管给予Nitrofen,正常对照组给食用油,孕21.5d时对所有孕鼠行剖宫产,取出胎鼠两侧肺组织进行EGF和EGFR免疫组化染色和图像分析。结果实验组死亡1只,致畸率46.7%;CDH肺发育不良,处于假腺体期和原始肺小管期,EGF表达上调而EGFR表达下调(P<0.05),膈疝侧与非膈疝侧肺组织EGF及EGFR表达差异没有统计学意义(P>0.05);实验组内产生CDH者胎肺内EGF和EGFR表达与无CDH者相差不大(P>0.05)。结论大鼠CDH模型中,腹腔内器官进入胸腔对肺组织的压迫可能并不是肺发育不良的主要原因,肺发育不良于膈疝形成前就已发生。CDH肺发育不良与EGF EGFR系统的变化密切相关。     

Pulmonary FGF-18 gene expression is downregulated during the canalicular-saccular stages in nitrofen-induced hypoplastic lungs

Purpose

Pulmonary hypoplasia (PH) associated with congenital diaphragmatic hernia (CDH) represents one of the major challenges in neonatal intensive care. However, the molecular pathogenesis of PH is still poorly understood. In developing fetal lungs, fibroblast growth factor 18 (FGF-18) plays a crucial role in distal airway maturation. FGF-18 knockouts show smaller lung sizes with reduced alveolar spaces and thicker interstitial mesenchymal compartments, highlighting its important function for fetal lung growth and differentiation. We hypothesized that pulmonary FGF-18 gene expression is downregulated during late gestation in nitrofen-induced hypoplastic lungs.

Methods

Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetuses were harvested on D18 and D21, and lungs were divided into three groups: controls, hypoplastic lungs without CDH [CDH(?)], and hypoplastic lungs with CDH [CDH(+)] (n = 24 at each time-point). Pulmonary FGF-18 gene expression levels were analyzed by qRT-PCR. Immunohistochemistry was performed to investigate FGF-18 protein expression/distribution.

Results

Relative mRNA levels of pulmonary FGF-18 gene expression were significantly decreased in CDH(?) and CDH(+) on D18 and D21 compared to controls (p < 0.05 and p < 0.01, respectively). Immunoreactivity of FGF-18 was markedly diminished in mesenchymal cells surrounding the airway epithelium on D18 and D21 compared to controls.

Conclusion

Downregulation of FGF-18 gene expression in nitrofen-induced hypoplastic lungs suggests that decreased FGF-18 expression during the canalicular–saccular stages may interfere with saccular–alveolar differentiation and distal airway maturation resulting in PH.     

Lung morphology after late fetal tracheal ligation in rats.

Pulmonary hypoplasia and persistent pulmonary hypertension are the main causes of mortality and morbidity in congenital diaphragmatic hernia (CDH). Prenatal tracheal occlusion accelerates lung growth, but the mechanism remains unknown. In order to be able to establish the accuracy of our experimental model for further molecular biological examinations, we evaluated the histologic structure of 1. fetal lungs subjected to tracheal occlusion compared to 2. normal fetal lungs, 3. hypoplastic lungs in CDH, and 4. normal neonatal lungs. One group of Sprague-Dawley rat fetuses were subjected to intrauterine tracheal ligation (TL) on gestational day 19 (n = 7). Control fetuses were obtained from the same litters as those subjected to TL (n = 8). Another group of pregnant Sprague-Dawley rats were given 100 mg nitrofen on gestational day 9.5 to create CDH (n = 8). All fetuses were delivered by cesarean section on day 21. Lungs from 1-day-old, healthy, non-operated, newborn Sprague-Dawley rats were also examined (n = 6). Lung weight to body weight ratio was significantly higher in the TL lungs (5.0 +/- 0.36 %), compared to control lungs (2.8 +/- 0.15 %), CDH lungs (1.9 +/- 0.12 %), and normal neonatal lungs (4.2 +/- 0.18 %). Volume density of alveolar air space and radial alveolar count (RAC) in TL lungs (52 +/- 1.4 %) (3.3 +/- 0.25) were significantly higher than in control lungs (34 +/- 3.4 %) (2.2 +/- 0.17) and in CDH lungs (16 +/- 1.7 %) (1.7 +/- 0.07). No significant differences were found between the TL and the normal neonatal group (59 +/- 1.4 %) (3.6 +/- 0.11). Fetal lungs after TL showed evidence of growth stimulation with increased volume density of alveolar air space and increased RAC, comparable to findings in normal neonatal lungs.     

Prenatal administration of retinoic acid increases the trophoblastic insulin-like growth factor 2 protein expression in the nitrofen model of congenital diaphragmatic hernia

Background

The high mortality rate in congenital diaphragmatic hernia (CDH) is attributed to pulmonary hypoplasia (PH). Insulin-like growth factor 2 (IGF2) is an important regulator of fetal growth. The highest levels of IGF2 expression are found in the placenta, which are negatively regulated by decidual retinoid acid receptor alpha (RARα). It has been demonstrated that prenatal administration of retinoic acid (RA) suppresses decidual RARα expression. Previous studies have further shown that prenatal administration of RA can reverse PH in nitrofen-induced CDH model. In IGF2 knockout animals, low levels of IGF2 are associated with decreased placental growth and PH. We therefore hypothesized that nitrofen decreases trophoblastic IGF2 expression and prenatal administration of RA increases it through decidual RARα in the nitrofen-induced CDH model.

Methods

Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). RA was given intraperitoneally on D18, D19 and D20. Fetuses were harvested on D21 and divided into three groups: control, CDH and nitrofen+RA. Immunohistochemistry was performed to evaluate decidual RARα and trophoblastic IGF2 expression. Protein levels of IGF2 in serum, intra-amniotic fluid and left lungs were measured by enzyme-linked immunosorbent assay.

Results

Significant growth retardation of placenta and left lungs was observed in the CDH group compared to control and nitrofen+RA group. Markedly increased decidual RARα and decreased IGF2 immunoreactivity were found in the CDH group compared to control and nitrofen+RA group. Significantly decreased IGF2 protein levels were detected in serum, intra-amniotic fluid and left lungs in the CDH group compared to control and nitrofen+RA group.

Conclusion

Our findings suggest that nitrofen may disturb trophoblastic IGF2 expression through decidual RARα resulting in retarded placental growth and PH in the nitrofen-induced CDH. Prenatal administration of RA may promote lung and placental growth by increasing trophoblastic IGF2 expression.     

Decreased pulmonary c-Cbl expression and tyrosine phosphorylation in the nitrofen-induced rat model of congenital diaphragmatic hernia

Purpose

The high morbidity of newborn infants with congenital diaphragmatic hernia (CDH) is attributed to pulmonary hypoplasia (PH), which is characterized by a failure of alveolar development. The nitrofen-induced CDH model has been widely used to investigate the pathogenesis of PH in CDH. It has previously been shown that the fibroblast growth factor receptor (FGFR) pathway, which is essential for a proper lung development, is disrupted during late gestation of nitrofen-induced CDH. Casitas B-lineage lymphoma (c-Cbl) proteins are known regulators of signal transduction through FGFRs, indicating their important role during alveolarization in developing lungs. Furthermore, it has been demonstrated that tyrosine phosphorylation of c-Cbl proteins has a pivotal role for their physiological function and activity during fetal lung development. We designed this study to test the hypothesis that pulmonary c-Cbl expression and tyrosine phosphorylation status are decreased in the nitrofen-induced CDH model.

Methods

Timed-pregnant rats received either 100 mg nitrofen or vehicle on gestation day 9 (D9). Fetuses were harvested on D18 and D21, and lungs were divided into two groups: control and hypoplastic lungs with CDH (CDH⁺) (n = 10 at each time-point, respectively). Pulmonary gene expression levels of c-Cbl were analyzed by quantitative real-time polymerase chain reaction. Western blotting combined with densitometry analysis was used for semi-quantification of protein levels of pulmonary c-Cbl and tyrosine phosphorylation status. Confocal-immunofluorescence staining was performed to evaluate c-Cbl protein expression and distribution.

Results

Relative mRNA expression levels of pulmonary c-Cbl were significantly decreased in CDH⁺ on D18 and D21 compared to controls. Western blotting showed markedly decreased protein levels of pulmonary c-Cbl and tyrosine phosphorylation status in CDH⁺ on D18 and D21. Confocal-immunofluorescence analysis confirmed decreased c-Cbl expression in CDH⁺ on D18 and D21 mainly in the distal alveolar epithelium compared to controls.

Conclusion

Decreased pulmonary c-Cbl gene and protein expression accompanied by a decreased tyrosine phosphorylation status during the late stages of fetal lung development may result in reduced c-Cbl activity, and thus interfere with the FGFR-mediated alveolarization in the nitrofen-induced CDH model.     

Downregulation of insulin-like growth factor binding protein 3 and 5 in nitrofen-induced pulmonary hypoplasia

Purpose

The high mortality in congenital diaphragmatic hernia (CDH) is mainly attributed to pulmonary hypoplasia. Recent studies suggest that retinoid signaling pathway (RSP) is inhibited in the nitrofen-induced hypoplastic lung. The insulin-like growth factor (IGF) system plays a crucial role in fetal lung development by interaction of IGFBP-3 and IGFBP-5 with RSP. We hypothesized that pulmonary IGFBP-3 and IGFBP-5 gene expression levels are downregulated in the nitrofen-induced pulmonary hypoplasia.

Methods

Pregnant rats were exposed to either olive oil or 100 mg nitrofen on day 9.5 (D9.5) of gestation. Fetal lungs were harvested on D18 and D21 and divided into control and nitrofen groups. IGFBP-3 and IGFBP-5 pulmonary gene and protein expression were determined using real-time RT–PCR and immunohistochemistry.

Results

Relative levels of IGFBP-3 mRNA were significantly decreased in the nitrofen group (8.00 ± 14.44) in D21 compared to controls (14.81 ± 16.11; p < 0.05). Expression levels of IGFBP-5 mRNA were also significantly decreased in nitrofen group (10.66 ± 4.83) on D18 compared to controls (17.92 ± 4.77). Immunohistochemistry showed decreased IGFBP-3 expression on D21 and decreased IGFBP-5 immunoreactivity on D18 in hypoplastic lungs compared to controls.

Conclusion

Downregulation of IGFBP-3 and IGFBP-5 gene expression may cause pulmonary hypoplasia in the nitrofen-induced CDH model by interfering with retinoid signaling pathway.     

Evidence for decreased lipofibroblast expression in hypoplastic rat lungs with congenital diaphragmatic hernia

Purpose

Pulmonary hypoplasia (PH) is a serious condition in newborns with congenital diaphragmatic hernia (CDH). Lipid-containing interstitial fibroblasts (LIFs) play an essential role in fetal lung maturation by stimulating alveolarization and lipid homeostasis. In rodents, LIFs are first evident during the canalicular phase of lung development with a significant increase over the last 4 days of gestation. Adipocyte differentiation-related protein (ADRP), a functional lipogenic molecular marker characterizing LIFs, is highly expressed in fetal lungs during this critical time period. We hypothesized that LIF expression in hypoplastic rat lungs is decreased in the nitrofen-induced CDH model, which is accompanied by reduced alveolar ADRP expression and lipid content.

Methods

On embryonic day 9.5 (E9.5), time-mated rats received either nitrofen or vehicle. Fetuses were sacrificed on selected time points E18.5 and E21.5, and dissected lungs were divided into controls and CDH-associated PH. Pulmonary gene expression levels of ADRP were determined by quantitative real-time polymerase chain reaction. ADRP immunohistochemistry and oil red O staining were used to assess pulmonary protein expression and lipid content. Immunofluorescence double staining for alpha smooth muscle actin, which is known to be absent in LIFs, and lipid droplets was performed to evaluate the pulmonary expression of this specific subset of fibroblasts.

Results

Relative mRNA expression of ADRP was significantly reduced in lungs of CDH-associated PH on E18.5 and E21.5 compared to controls. ADRP immunoreactivity and lipid staining were markedly diminished in alveolar mesenchymal cells of CDH-associated PH on E18.5 and E21.5 compared to controls. Confocal laser scanning microscopy demonstrated markedly decreased LIF expression in alveolar interstitium of CDH-associated PH on E18.5 and E21.5 compared to controls.

Conclusion

Decreased pulmonary LIF expression during late gestation suggests impaired LIF functioning in the nitrofen-induced CDH model, which may cause disruption in fetal alveolarization and lipid homeostasis, and thus contribute to the development of PH.