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.     

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.     

Hippo通路相关分子在先天性膈疝肺发育不良大鼠模型中的表达及意义

目的了解Hippo信号通路主要组成分子在先天性膈疝(congenital diaphragmatic hernia,CDH)模型大鼠胎肺组织中的表达情况,初步探讨该通路与CDH肺发育不良的关系。方法应用除草醚建立CDH大鼠模型,选取模型组中单纯左侧膈疝的胎鼠左肺作为研究对象,通过免疫印迹法和RT-PCR分别检测肺组织中Lats1、Taz和Yap(pYap)表达情况并与对照组胎鼠的左肺组织进行比较。结果 Lats1、Taz和Yap在模型胎鼠和正常胎鼠的支气管、肺上皮细胞及肺动脉壁中都有表达。模型组的Yap蛋白及其mRNA的表达量分别为2.333 3±0.367 5和1.243 3±0.103 3明显高于对照组的1.250 9±0.388 6和0.983 3±0.065 0,两组比较,差异有统计学意义(P<0.05);而Lats1蛋白及其mRNA和Yap蛋白磷酸化水平(pYap)的表达量在两组间的差异均无统计学意义(P>0.05)。Taz的mRNA表达量在模型组和对照组分别为1.216 7±0.033 9和1.013 3±0.183 4,组间比较,差异有统计学意义(P<0...     

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。     

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。     

成纤维细胞生长因子-7在除草醚诱导的先天性膈疝大鼠模型胎肺中的表达

目的研究成纤维细胞生长因子-7（FGF-7）在先天性膈疝（CDH）胎肺中的表达及探讨其在CDH肺发育不良中的作用。方法采用免疫组织化学和图像分析方法半定量地检测FGF-7在胎肺中的表达部位及表达相对含量。结果CDH肺发育不良，处于假腺体期。FGF-7在对照组显示强表达于支气管和细支气管上皮细胞，在除草醚（nitrofen）组中表达微弱，其表达的相对含量显著低于对照组，Nitrofen组中CDH组和noCDH组间差异没有显著性意义。FGF-7的表达与肺泡面积呈显著正相关性，与肺泡间隔呈显著负相关性。结论FGF-7在CDH胎肺中表达降低，且降低具有组织特异性；FGF-7可作为提示CDH胎肺成熟度的重要指标；FGF-7表达降低可能是CDH肺发育不良形成机制之一。     

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。     

高体积分数氧暴露对早产大鼠肺p53和细胞周期调节基因表达的影响

目的 观察体积分数为600 mL/L氧暴露对早产大鼠肺肿瘤抑制蛋白p53和肺细胞周期调节基因(p21waf/cipl)表达的影响,探讨其与新型支气管肺发育不良(BPD)发病机制的关系.方法 孕21 d早产大鼠出生6 h内随机分为高体积分数氧(高氧)组和对照组.对照组置于常压空气中,高氧组置于氧体积分数为600 mL/L的氧舱中,二组均于胚胎19 d(E19)及出生第1、3、5、7天(P1、P3、P5、P7)各随机取8只早产大鼠,采用RT-PCR技术检测其肺组织肺p53和p21waf/cipl基因表达水平.采用SPSS 12.0软件进行统计学分析.结果 1.对照组胎鼠及早产大鼠肺组织p53 mRNA的表达自出生后随鼠龄增加逐渐下降,至P5最低;高氧组胎鼠及早产大鼠p53 mRNA的表达在E19～P7均较高于对照组,P5和P7时与对照组比较差异均有统计学意义(Pa<0.05).2.对照组胎鼠及早产大鼠肺组织p21waf/cipl mRNA的表达水平在E19～P7随鼠龄增加而升高,高氧组胎鼠及早产大鼠肺组织p21waf/cipl mRNA的表达在E19～P7均高于对照组,P5时二组比较差异有统计学意义(P<0.05).结论 600 mL/L氧暴露可通过调控p53及p21waf/cipl途径抑制肺组织细胞增殖,进而导致BPD的发生.     

汉防己甲素对先天性膈疝大鼠模型胎仔肺发育不良的影响及意义

目的 探讨产前应用汉防己甲素(TET)对先天性膈疝(CDH)大鼠模型胎仔肺内表面活性蛋白B(SP-B)和增殖细胞核抗原(PCNA)的影响及意义.方法 10只成年雌性SD大鼠配种后,于孕9.5 d随机分为3组:对照组(2只)、膈疝组(4只)和治疗组(4只),膈疝组和治疗组一次性灌胃给予除草醚125 mg/只(溶于2.5ml橄榄油中),对照组灌胃给予等量橄榄油.配种后18.5 d时,治疗组给予30 mg/kg汉防己甲素灌胃(1次/d,连续3 d),膈疝组和对照组给予等量生理盐水.孕21 d对孕鼠行剖宫产,观察胎鼠膈疝形成情况,取出肺组织行HE染色并行图像分析,采用免疫组化染色法检测胎肺内SP-B和PCNA的表达情况.结果膈疝组和治疗组共有48只胎鼠形成膈疝,其致畸率为64.9%,二组之间致畸率差别无统计学意义(P>0.05).膈疝组胎鼠存在肺发育不良,产前给予TET干预,肺组织在形态上有明显改善,外形接近于对照组.正常肺组织中,SP-B在肺泡上皮细胞及远端支气管上皮细胞的胞浆内表达,在膈疝组中未见SP-B表达.治疗组中可见SP-B表达,但低于对照组(P<0.01).PCNA阳性细胞率在对照组与膈疝组的差异无统计学意义(P>0.05),但在治疗组中其表达显著较前二组为低,差异有统计学意义(P<0.01).结论 产前应用TET可诱导CDH胎鼠肺的成熟,促进肺内SP-B表达升高;同时,产前给予TET干预的CDH胎鼠,其肺内PCNA阳性细胞率明显降低,提示TET诱导CDH胎肺的成熟并不是依靠促进细胞增殖来完成的.     

宫内炎性暴露对胎鼠及早产大鼠肺组织细胞增殖和p53基因表达的影响

目的 观察宫内炎性暴露对胎鼠及早产大鼠肺组织细胞增殖和p53基因表达的影响,探讨其与新型支气管肺发育不良(BPD)发病的关系.方法 定期受孕的SD大鼠随机分为脂多糖(LPS)组和对照组.分别于孕第15天用微量加样器在其羊膜腔内注射LPS(40 ug/L)5uL和等最无菌9 g/L盐水.二组均于胚胎19 d及出生第1、3、5、7天(E19、P1、P3、P5、P7)各随机取8只,采用免疫组织化学方法和反转录-聚合酶链反应技术检测肺组织细胞增殖性核抗原(PCNA)蛋白及p53 mRNA表达水平.应用SPSS12.0软件进行统计学分析.结果 对照组PCNA的表达在E19-P1随鼠龄增加表达逐渐减弱,至P5表达最弱,以后随鼠龄增加逐渐增多.LPS组PCNA在E19～P1随鼠龄增加逐渐减弱,至P1最弱,以后随鼠龄增加而逐渐升高,LPS组PCNA在E19～P3表达均低于对照组,差异具有统计学意义(Pa<0.05).对照组053 mRNA表达,在E19～P5随鼠龄增加逐渐下降,以后逐渐上升;LPS组p53 mRNA E19～P7均较对照组高,在E19～P1与对照组比较,差异有统计学意义(Pa<0.05).结论 宫内炎性暴露可能通过上调p53基因的表达,抑制胎鼠及早产大鼠肺组织细胞增殖,干扰正常的肺发育进程,进而导致BPD的发生.     

Effect of dexamethasone on endothelial nitric oxide synthase in experimental congenital diaphragmatic hernia

AIMS—To study the effect of prenatal glucocorticoid treatment on endothelial nitric oxide synthase (eNOS) expression in rats with congenital diaphragmatic hernia (CDH).METHODS—CDH was induced in fetal rats by the maternal administration of nitrofen on day 9.5 of gestation. Dexamethasone was administered on days 18.5 and 19.5 before delivery of the fetuses on days 20.5and 21.5. Pulmonary eNOS protein expression was studied by western immunoblotting and immunohistochemistry.RESULTS—On day 20.5, eNOS expression was significantly reduced in CDH pups compared with normal control rats. Dexamethasone treated CDH pups had eNOS concentrations equivalent to those of normal animals. By day 21.5, however, there was no detectable difference in eNOS expression between the experimental groups.CONCLUSIONS—eNOS is deficient in near term (day 20.5) CDH rats. Dexamethasone restores eNOS expression in these animals to that seen in normal rat lungs. At term, the precise role of eNOS in the pathophysiology of CDH remains uncertain.     

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.     

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.     

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.