维甲酸导致胎鼠神经脊源性器官异常

为证实我们的假设，即维甲酸物质不但能导致胎鼠颅面部的畸形，而且还能造成胸颈部内分泌腺(胸腺、甲状腺和甲状旁腺)的异常，这些器官的形成都是与神经脊细胞的移入有关的。于孕期第10天给母鼠125mg/kg全反型维甲酸(All-franretinoic acid)，对照组给予相应量的载体溶液。分娩前24小时剖腹取出胎鼠，10％福尔马林漫泡固定10天，双管手术显微镜下解剖，检查颅、面部，心脏，大血管，胸腺，并对甲状腺，甲状旁腺进行组织学研究。对照组10只胎鼠未发现畸形，而实验组20只胎鼠中，有17只(85％)出现颅面部畸形，14只(70％)有心血管畸形，而胸腺、甲状腺、甲状旁腺和甲状腺畸形分别有14只(70％)、15只(75％)和7只(35％)。结果表明，维甲酸能导致胎鼠中鳃弓衍化而成并需神经脊参与的器官组织结构异常。     

维甲酸导致胎鼠神经脊源性器官异常

为证实我们的假设,即维甲酸物质不但能导致胎鼠颅面部的畸形,而且还能造成胸颈部内分泌腺(胸腺、甲状腺和甲状旁腺)的异常,这些器官的形成都是与神经脊细胞的移入有关的.于孕期第10天给母鼠125mg/kg全反型维甲酸(All-franretinoicacid),对照组给予相应量的载体溶液.分娩前24小时剖腹取出胎鼠,10%福尔马林浸泡固定10天,双管手术显微镜下解剖,检查颅、面部,心脏,大血管,胸腺,并对甲状腺,甲状旁腺进行组织学研究.对照组10只胎鼠未发现畸形,而实验组20只胎鼠中,有17只(85%)出现颅面部畸形,14只(70%)有心血管畸形,而胸腺、甲状腺、甲状旁腺和甲状腺畸形分别有14只(70%)、15只(75%)和7只(35%).结果表明,维甲酸能导致胎鼠中鳃弓衍化而成并需神经脊参与的器官组织结构异常.     

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。     

地塞米松对先天性膈疝胎鼠肺发育的影响

目的研究地塞米松对先天性膈疝(Congenital Diaphragmatic Hernia,CDH)胎鼠肺发育的影响,并探讨其可能机制。方法采用Nitrofen法制作胎鼠CDH模型并分组,地塞米松组予以产前地塞米松治疗,Nitrofen组则不予地塞米松治疗,另取正常胎鼠为对照组;采用组织学检查方法评价肺发育情况,免疫组化和图像分析方法检测地塞米松作用前后胰岛素样生长因子-Ⅰ(Insulin-like Growth Factor,IGF-Ⅰ)在胎肺中的表达及相对含量。结果与Nitrofen组相比,地塞米松组肺组织中IGF-Ⅰ的表达明显降低,且肺组织发育明显改善。结论胎儿期地塞米松通过下调肺组织中IGF-Ⅰ的表达,来改善肺组织的发育。     

先天性膈疝动物模型制作及其肺部一氧化氮的改变

用Nitrofen致畸孕鼠，获取先天性膈疝鼠仔模型。共获得28只先天性膈疝（CDH）鼠仔，经Nitrofen处理未患CDH鼠仔34只，正常对照组鼠仔25只，对3组进行肺重/体重指数，肺蛋白定量、肺中一氧化氮合成酶（NOS）和一氧化氮（NO）研究。结果发现用Nitrofen致畸孕鼠，在鼠仔中能产生CDH；病侧肺组织中NO含量下降，NOS酶活性下降。由于NO能舒张血管平滑肌细胞。因此病侧肺组织中NO含量明显下降。和CDH肺高压病理生理有关。NO制剂量治疗CDH肺高压一种有希望的新途径。     

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.     

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。     

先天性膈疝对肺发育的影响及其防治措施

先天性膈疝(Congenital Diaphragmatic Hernia，CDH)是先天隔肌发育不良而导致的畸形，腹部脏器经膈肌缺损疝人胸腔，引起一系列病理生理变化，对胎儿或患儿的心肺发育、心肺功能均造成了不同程度的影响。国外资料表明，其发病率约为1：2000～1：5000。患儿出生后，由于心肺发育不全，病死率较高。近年来，对CDH所致的肺发育不     

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。     

Neural crest-derived defects in experimental congenital diaphragmatic hernia

 Congenital diaphragmatic hernia (CDH) is often associated with other malformations. This study tests the hypothesis that the heart and great vessels, thymus, parathyroids, and thyroid might be abnormal in the rat model of CDH as a result of disturbed neural-crest development. Time-mated pregnant rats were fed either 100 mg 2-4-dichlorophenyl-p-nitrophenyl ether (nitrofen) or vehicle on gestational day 9.5. Diaphragm, lung, heart, and thymic malformations were sought after dissection and the parathyroids and thyroid were histologically investigated in term fetuses. Ten control fetuses had no malformations, whereas 22 of 32 nitrofen fetuses had CDH and 20 had cardiovascular defects like narrow pulmonary outflow tract (n=7), aberrant right subclavian artery (n=7), ventricular septal defect (n=4), atrial septal defect (n=4), tetralogy of Fallot (n=2), double-outflow right ventricle (n=2), right ductus arteriosus (n=2), and others. The thymus was present but was significantly hypoplastic in all nitrofen fetuses and was ectopic or single-lobed in 28% of them while the parathyroid glands were unilaterally absent or ectopic in 50%. The thyroid was only minimally malformed or ectopic. In conclusion, malformations of structures derived from the pharyngeal arches are likely neural-crest related in rats exposed to nitrofen.     

Effects of early embryonal exposure to dexamethasone on malformations of neural-crest derivatives induced by nitrofen in rats

Prenatal corticosteroids reverse to some extent lung and heart hypoplasia in nitrofen-exposed rat pups. The present study examines the effects of early exposure to dexamethasone on the neural crest-related malformations of the cardiovascular system, thymus, parathyroids, and thyroid observed in this model. Pregnant rats were exposed on gestational day 9.5 to either 100 mg 2-4-dichlorophenyl-p-nitrophenyl ether (nitrofen) alone or followed on days 10.5 and 11.5 by 0.4 mg/kg dexamethasone (dexa) i.p. Controls were treated with either oil alone or oil+dexa alone. The fetuses were recovered near term and diaphragmatic, lung, heart, and thymic malformations were sought after dissection. The parathyroids and thyroid were histologically investigated. Control fetuses had no malformations whereas 68% of nitrofen and 65% of nitrofen + dexa fetuses had congenital diaphragmatic hernias (CDH). Heart-outflow tract and pharyngeal artery anomalies were seen in 62% and 61%, respectively in both groups. Heart hypoplasia, which was severe in the nitrofen group, was fully reversed in nitrofen+dexa pups. In contrast, thymic hypoplasia was of similar severity in both groups. The hypoplastic thymus was malformed in 29% and 39%, the parathyroids in 50% and 41%, and the thyroid in 25% and 16% of fetuses, respectively. These differences were not significant. Early exposure to dexa in rat fetuses previously treated with nitrofen thus does not produce any benefit on the incidence or severity of malformations of the cardiac outflow tract and pharyngeal derivatives that accompany CDH in rats exposed to nitrofen. However, even administered so early, this medication prevents heart hypoplasia, suggesting a favorable effect on early heart organogenesis.     

Effects of vitamin A on malformations of neural-crest-controlled organs induced by nitrofen in rats

Vitamin A (vit A) alleviates the effects of nitrofen in exposed rat pups. The present study examines the effects of early exposure to vitamin A on the neural-crest-related cardiovascular, thymic, parathyroid, and thyroid malformations previously reported in the rat model of congenital diaphragmatic hernia (CDH). Pregnant rats were exposed on gestational day 9.5 to 100 mg 2-4-dichlorophenyl-p-nitrophenyl ether (nitrofen) alone or followed by 15,000 IU vit A. Controls were treated only with oil or oil + vit A. The fetuses were recovered near term and diaphragmatic, lung, heart, and thymic malformations were sought after dissection. The parathyroids and thyroid were histologically investigated. The hearts were also examined for protein, DNA, and proportion of proliferating cells. None of the control fetuses had malformations, whereas 41% of nitrofen and 27% of nitrofen + vit A fetuses had CDH. Anomalies of the heart outflow tract and pharyngeal arteries were seen in 64% and 43%, respectively, in both groups. Heart and thymic hypoplasia, which were severe in the nitrofen group with significant decreases of total DNA and percent proliferating cells, were significantly improved in the nitrofen + vit A group. The hypoplastic thymus was malformed in 53% and 27% of fetuses, respectively, and the parathyroids were abnormal in 48% and 35%, respectively. Only minimal anomalies of the thyroid were found. The significant improvement of heart and thymic hypoplasia associated with vit A was not seen for the other variables studied, but there was a trend in this direction for all of them. Vit A definitely improved heart hypoplasia induced by nitrofen by stimulating myogenesis. It also improved thymic hypoplasia, but had limited beneficial effects on malformations of the cardiac outflow tract and pharyngeal derivatives that accompany CDH in rats exposed to nitrofen.     

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.     

Neural crest-derived defects in experimental esophageal atresia

Esophageal atresia (EA) is often associated with cardiovascular and other malformations that are likely neural crest derived. The present study tests the hypothesis that the heart and great vessels and the thymus and parathyroids may be abnormal in the rat model of EA as a result of disturbed neural crest development. Time-mated pregnant rats received intraperitoneally on d 8 and 9 of gestation either 2 mg/kg adriamycin or vehicle. Esophageal, heart, and thymic malformations were sought under the microscope in term fetuses. The parathyroids were histologically investigated. Control fetuses had no malformations, whereas 69 of 109 fetuses exposed to adriamycin had EA and 45 of 69 had 15 right aortic arches, nine aberrant right subclavia, eight ventricular septal defects, six narrow pulmonary outflow tracts, five tetralogies of Fallot, three double outflow right ventricles, three double aortic arches, three atrial septal defects, three right ductus arteriosus, and two truncus. The thymus was absent in 19, hypoplastic in 12, and ectopic in five out of 36 fetuses with EA in which it was studied, whereas the parathyroid glands were absent in 16, single in four, and ectopic in one of the 23 fetuses with EA in which they were studied. In conclusion, the nature of the cardiovascular, thymic, and parathyroid malformations associated with EA in rats is consistent with the hypothesis of neural crest participation in their pathogenesis. Mechanisms simultaneously disturbing foregut septation, somitic segmentation, and neural crest development should be sought to explain the combined occurrence of malformations in EA.     

Spatial and temporal expression of glucocorticoid, retinoid, and thyroid hormone receptors is not altered in lungs of congenital diaphragmatic hernia

The degree of associated pulmonary hypoplasia and persistent pulmonary hypertension are major determination factors for survival in congenital diaphragmatic hernia (CDH) patients. Glucocorticoids, thyroid hormone, and vitamin A have been shown to be involved in human lung development. To determine their therapeutic potential in hypoplastic lungs of CDH patients, the temporal and spatial expression of glucocorticoid receptor, thyroid hormone receptors, retinoic acid receptors, and retinoid X receptors were evaluated in lungs of CDH patients, hypoplastic lungs from other causes, and normal lungs. As a series of supportive experiments, the expressions of these receptors were analyzed in lungs of nitrofen-induced CDH rats. Immunohistochemistry (human and rat) and in situ hybridization (rat) demonstrated no overt difference between CDH, hypoplastic, and control lungs, either in the localization nor the timing of the first expression of all analyzed receptors. The mRNA expression of each receptor was detected in all human CDH lungs by quantitative PCR. Our results suggest that, as far as receptors are concerned, hypoplastic lungs of fetuses and newborns with CDH are potentially as responsive to glucocorticoids, thyroid hormone, and retinoic acid as the lungs of normal children.     

Pathogenesis of a Bisdiamine-Induced Malformation Complex in Rat Resembling DiGeorge Syndrome

Abstract Administration of the antispermatogenic agent bis-diamine (N, N'-bis-(dichloroacetyl)-1, 8-octamethylenediamine) to pregnant rats produced a malformation complex resembling DiGeorge syndrome in man. The malformations consisted of hypoplasia or aplasia of the thymus, persistent truncus arteriosus, tetralogy of Fallot, aberrant subclavian artery and other cardiovascular anomalies, as well as hypoplasia of the parathyroid gland, thyroid gland, and the spleen. The malformation complex appeared with a high incidence when the drug was administered on day 9.5 or day 10 of gestation.
Since the connective tissues of the thymus, aorticopulmonary septum, smooth muscles of the media of the aortic arch artery, and connective tissues of the parathyroid and the thyroid glands, are of neural crest origin, and since the time of their migration is around day 10 of gestation, we postulate that the action of bis-diamine might be involution of the migration of the neural crest cells and of its pathways.
We further suggest from the findings of persistent atrioventricular canal and hypoplasia of the spleen, that the drug must have also affected the growth of mesenchymal cells, which originate from the endocardial epithelial cells.     

The relationship of diaphragmatic defect,liver growth,and lung hypoplasia in nitrofen-induced congenital diaphragmatic hernia in the rat

Reduced lung size (lung hypoplasia, LH) is the main cause of mortality in newborns with congenital diaphragmatic hernia (CDH). However, it is unclear which mechanisms lead to LH. To assess this, we analyzed the relationship of LH and liver mass in correlation to the size of the diaphragmatic defect in rats with nitrofen-induced CDH. A total of 266 newborn Sprague-Dawley rats (30 litters) were exposed to nitrofen on day 11.5 of pregnancy. After spontaneous delivery at term (22 days), all newborns were microdissected. Using a computerized morphometric device, the area of the thoracic cavity, the lung, the intrathoracic liver, and the diaphragmatic defect were measured. The lungs, the intrathoracic, and the extrathoracic portion of the liver were weighed. After nitrofen exposure, 160 newborn rats presented with CDH (60.2%). They were divided into five groups according to the intrathoracic content of intraabdominal organs. We observed a significant increase of the total liver and decrease of the lung weight in the severely affected groups. A significant correlation between the size of the defect and the weight of the intrathoracic part of the liver could be demonstrated. Nitrofen alone had no effect on liver weight. Our results indicate that (1) the presence of liver inside the thoracic cavity is not the result of dislocation but rather of growth of liver tissue through the defect, and (2) the observed correlation between the size of the defect and the intrathoracic liver weight may be part of the pathogenesis of LH in CDH.