Takahashi Toshiaki, Friedmacher Florian, Zimmer Julia, Puri Prem
National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland.
Conway Institute of Biomolecular and Biomedical Research, School of Medicine and Medical Science, University College Dublin, Dublin, Ireland.
Pediatr Surg Int. 2016 Dec;32(12):1127-1132. doi: 10.1007/s00383-016-3968-0. Epub 2016 Sep 20.
Congenital diaphragmatic hernia (CDH) is presumed to originate from defects in the primordial diaphragmatic mesenchyme, mainly comprising of muscle connective tissue (MCT). Thus, normal diaphragmatic morphogenesis depends on the structural integrity of the underlying MCT. Developmental mutations that inhibit normal formation of diaphragmatic MCT have been shown to result in CDH. Desmin (DES) is a major filament protein in the MCT, which is essential for the tensile strength of the developing diaphragm muscle. DES knockout mice exhibit significant reductions in stiffness and elasticity of the developing diaphragmatic muscle tissue. Furthermore, sequence changes in the DES gene have recently been identified in human cases of CDH, suggesting that alterations in DES expression may lead to diaphragmatic defects. This study was designed to investigate the hypothesis that diaphragmatic DES expression is decreased in fetal rats with nitrofen-induced CDH.
Time-mated Sprague-Dawley rats were exposed to either nitrofen or vehicle on gestational day 9 (D9). Fetuses were harvested on selected time-points D13, D15 and D18, and dissected diaphragms (n = 72) were divided into control and nitrofen-exposed specimens (n = 12 per time-point and experimental group, respectively). Laser-capture microdissection was used to obtain diaphragmatic tissue elements. Diaphragmatic gene expression of DES was analyzed by quantitative real-time polymerase chain reaction. Immunofluorescence double staining for DES was combined with the mesenchymal marker GATA4 to evaluate protein expression and localization in developing fetal diaphragms.
Relative mRNA expression levels of DES were significantly decreased in pleuroperitoneal folds on D13 (1.49 ± 1.79 vs. 3.47 ± 2.32; p < 0.05), developing diaphragms on D15 (1.49 ± 1.41 vs. 3.94 ± 3.06; p < 0.05) and fully muscularized diaphragms on D18 (2.45 ± 1.47 vs. 5.12 ± 3.37; p < 0.05) of nitrofen-exposed fetuses compared to controls. Confocal laser scanning microscopy demonstrated markedly diminished immunofluorescence of DES mainly in diaphragmatic MCT, which was associated with a reduction of proliferating mesenchymal cells in nitrofen-exposed fetuses on D13, D15 and D18 compared to controls.
Decreased expression of DES in the fetal diaphragm may disturb the basic integrity of myofibrils and the cytoskeletal network during myogenesis, causing malformed MCT and leading to diaphragmatic defects in the nitrofen-induced CDH model.
先天性膈疝（CDH）被认为起源于原始膈间充质的缺陷，主要由肌肉结缔组织（MCT）组成。因此，正常的膈形态发生依赖于其下方MCT的结构完整性。已表明抑制膈MCT正常形成的发育突变会导致CDH。结蛋白（DES）是MCT中的一种主要细丝蛋白，对发育中的膈肌的抗张强度至关重要。DES基因敲除小鼠的发育中的膈肌组织的刚度和弹性显著降低。此外，最近在人类CDH病例中发现了DES基因的序列变化，这表明DES表达的改变可能导致膈缺陷。本研究旨在探讨在硝呋烯腙诱导的CDH胎鼠中膈DES表达降低这一假说。
将定时交配的Sprague-Dawley大鼠在妊娠第9天（D9）暴露于硝呋烯腙或溶剂。在选定的时间点D13、D15和D18收集胎儿，并将解剖后的膈肌（n = 72）分为对照组和硝呋烯腙暴露组标本（每个时间点和实验组分别为n = 12）。使用激光捕获显微切割技术获取膈组织样本。通过定量实时聚合酶链反应分析DES的膈基因表达。DES免疫荧光双染色与间充质标志物GATA4相结合，以评估发育中的胎儿膈肌中蛋白质的表达和定位。
与对照组相比，在D13时硝呋烯腙暴露胎儿的胸腹皱襞中DES的相对mRNA表达水平显著降低（1.49±1.79对3.47±2.3；p<0.05），在D15时发育中的膈肌中（1.49±1.41对3.94±3.06；p<0.05），以及在D18时完全肌化的膈肌中（2.45±1.47对5.12±3.37；p<0.05）。共聚焦激光扫描显微镜显示，DES的免疫荧光主要在膈MCT中明显减弱，这与D13、D15和D18时硝呋烯腙暴露胎儿中增殖间充质细胞的减少有关，与对照组相比。
胎儿膈肌中DES表达降低可能会在肌生成过程中干扰肌原纤维和细胞骨架网络的基本完整性，导致MCT畸形，并在硝呋烯腙诱导的CDH模型中导致膈缺陷。
