Dingemann J, Doi T, Ruttenstock E M, Gosemann J H, Puri P
National Children's Research Centre, Our Lady's Children's Hospital, Dublin, Ireland.
Eur J Pediatr Surg. 2012 Feb;22(1):21-5. doi: 10.1055/s-0031-1284358. Epub 2011 Aug 30.
The nitrofen model of congenital diaphragmatic hernia (CDH) creates a Bochdalek-type diaphragmatic defect and has been widely used to investigate the pathogenesis of CDH. However, the exact pathogenesis of the diaphragmatic defect in this model is still poorly understood. Chicken ovalbumin upstream promotor-transcription factor II (COUP-TFII) is expressed in the embryonic pleuroperitoneal folds (PPF) in the early stage of development and in the diaphragm in the late days of gestation. COUP-TFII is known to be a strong repressor of the retinoid signaling pathway (RSP), which plays an important role in diaphragm development. Furthermore, it has been recently shown that COUP-TFII is upregulated during early gestation in the nitrofen-induced hypoplastic lung. We designed this study to investigate the hypothesis that COUP-TFII gene expression is upregulated during early diaphragmatic development in the PPF.
Timed pregnant rats were exposed to either olive oil (Control) or nitrofen (CDH) on day 9 of gestation (D9). Fetuses were sacrificed on D13, D18 or D21. The PPF was dissected from D13 fetuses using laser capture microdissection. Diaphragms were dissected from D18 and D21 fetuses under the dissection microscope. The relative mRNA expression levels of COUP-TFII were determined using real-time PCR. Immunohistochemistry was performed to evaluate diaphragmatic protein expression and the distribution of COUP-TFII.Results On D13, gene expression levels of COUP-TFII in the PPF were significantly increased in the CDH group (82.93 ± 11.85) compared to Controls (46.22 ± 8.09; p < 0.05), whereas there were no differences at later time points. The immunoreactivity of diaphragmatic COUP-TFII was markedly increased in the PPF in the CDH group compared to controls on D13. No difference in immunoreactivity was observed on D18 and D21.
Upregulation of COUP-II gene expression in the PPF may contribute to the diaphragmatic defect in the nitrofen CDH model by inhibiting the RSP.
先天性膈疝（CDH）的硝基芬模型会造成Bochdalek型膈肌缺损，已被广泛用于研究CDH的发病机制。然而，该模型中膈肌缺损的确切发病机制仍知之甚少。鸡卵清蛋白上游启动子转录因子II（COUP-TFII）在发育早期的胚胎胸腹皱襞（PPF）以及妊娠后期的膈肌中表达。已知COUP-TFII是视黄酸信号通路（RSP）的强效抑制剂，而RSP在膈肌发育中起重要作用。此外，最近研究表明，在硝基芬诱导的肺发育不全的妊娠早期，COUP-TFII表达上调。我们设计本研究以探讨PPF中膈肌早期发育过程中COUP-TFII基因表达上调这一假说。
在妊娠第9天（D9）给定时怀孕的大鼠分别注射橄榄油（对照组）或硝基芬（CDH组）。在D13、D18或D21处死胎儿。使用激光捕获显微切割技术从D13胎儿中分离出PPF。在解剖显微镜下从D18和D21胎儿中分离出膈肌。使用实时PCR测定COUP-TFII的相对mRNA表达水平。进行免疫组织化学以评估膈肌蛋白表达及COUP-TFII的分布。
在D13时，与对照组（46.22±8.09）相比，CDH组PPF中COUP-TFII的基因表达水平显著升高（82.93±11.85；p<0.05），而在后期时间点无差异。与对照组相比，CDH组D13时PPF中膈肌COUP-TFII的免疫反应性显著增加。在D18和D21时未观察到免疫反应性差异。
PPF中COUP-II基因表达上调可能通过抑制RSP导致硝基芬CDH模型中的膈肌缺损。
