Dingemann Jens, Doi Takashi, Ruttenstock Elke, Puri Prem
National Children's Research Centre, Our Lady's Children's Hospital Dublin, Ireland University College Dublin, Dublin 12, Ireland.
Pediatr Surg Int. 2011 Feb;27(2):159-63. doi: 10.1007/s00383-010-2795-y.
The nitrofen model of congenital diaphragmatic hernia (CDH) reproduces a typical diaphragmatic defect. However, the exact pathomechanism of CDH is still unknown. The Wilm's tumor 1 gene (WT1) is crucial for diaphragmatic development. Mutations in WT1 associated with CDH have been described in humans. Additionally, WT1(-/-) mice display CDH. Furthermore, WT1 is involved in the retinoid signaling pathway, a candidate pathway for CDH. We hypothesized that diaphragmatic WT1 gene expression is downregulated during diaphragmatic development in the nitrofen CDH model.
Pregnant rats received vehicle or nitrofen on gestational day 9 (D9). Embryos were delivered on D13, D18 and D21. The pleuroperitoneal folds (PPFs) were dissected using laser capture microdissection (D13). Diaphragms of D18 and D21 were manually dissected. RNA was extracted and relative mRNA expression of WT1 was determined using real-time PCR. Immunofluorescence was performed to evaluate protein expression of WT1. Statistical significance was considered p < 0.05.
Diaphragmatic mRNA expression of WT1 was significantly decreased in the nitrofen group on D13, D18 and D21. Intensity of immunofluorescencence of WT1 was markedly decreased in the CDH diaphragms on D13, D18 and D21.
Downregulation of diaphragmatic WT1 gene expression may impair diaphragmatic development in the nitrofen CDH model.
先天性膈疝（CDH）的硝苯呋海因模型可再现典型的膈肌缺损。然而，CDH的确切发病机制仍不清楚。威尔姆斯瘤1基因（WT1）对膈肌发育至关重要。人类中已描述了与CDH相关的WT1突变。此外，WT1基因敲除（WT1-/-）小鼠表现出CDH。此外，WT1参与视黄酸信号通路，这是CDH的一个候选通路。我们假设在硝苯呋海因CDH模型中，膈肌发育过程中WT1基因表达下调。
妊娠大鼠在妊娠第9天（D9）接受载体或硝苯呋海因处理。分别在D13、D18和D21分娩胚胎。在D13使用激光捕获显微切割技术分离胸腹皱襞（PPF）。在D18和D21手动分离膈肌。提取RNA，并使用实时PCR测定WT1的相对mRNA表达。进行免疫荧光以评估WT1的蛋白表达。统计学显著性以p < 0.05为标准。
在D13、D18和D21，硝苯呋海因组膈肌WT1的mRNA表达显著降低。在D13、D18和D21，CDH膈肌中WT1的免疫荧光强度明显降低。
在硝苯呋海因CDH模型中，膈肌WT1基因表达下调可能会损害膈肌发育。
