Lukošiūtė A, Doi T, Dingemann J, Ruttenstock E M, Puri P
National Children's Research Centre, Our Lady's Children's Hospital, Dublin, Ireland.
Eur J Pediatr Surg. 2011 Jan;21(1):38-41. doi: 10.1055/s-0030-1262800. Epub 2010 Nov 4.
Pulmonary hypoplasia is a primary cause of high morbidity and mortality in neonates with Congenital Diaphragmatic Hernia (CDH). However, the precise pathogenesis of PH associated with CDH is still not clearly understood. It has been recently reported that lung Kruppel-like factor (LKLF), a member of the Kruppel-like factor family of transcription factors, is predominantly expressed in lungs and plays an important role in lung morphogenesis and functional maturation. It has been reported that homozygous deletion of LKLF gene in mice results in reduced lung morphogenesis. It is further reported that chimeric mice derived from LKLF (-/-) embryonic stem cells exhibit delayed lung development especially in the later gestational stages. We therefore designed this study to test the hypothesis that the LKLF gene is down-regulated during later stages of lung development in nitrofen-induced hypoplastic lungs.
Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetal lungs were harvested on D15, D18, and D21 and divided into 3 groups:control, nitrofen without CDH(CDH(-)) and nitrofen with CDH(CDH(+)) (n=24 for each group). Real-time RT-PCR analysis was performed to investigate pulmonary gene expression levels of LKLF. Differences between the 3 groups at each time point were tested statistically and significance was accepted at p<0.05. Immunohistochemistry was also performed to evaluate LKLF protein expression and distribution.
The relative mRNA expression levels of LKLF on D18 and D21 were significantly decreased (p<0.01) in CDH(-) and CDH(+) groups compared to controls. The gene expression levels of LKLF on D15 did not differ significantly between the nitrofen group and controls. Immunohistochemical study showed strong LKLF immunoreactivity on D18 and D21 in nitrofen-induced hypoplastic lung compared to controls, whereas no difference was seen on D15.
Our results provide evidence for the first time that LKLF is down-regulated in the later stages of lung development in nitrofen-induced hypoplastic lungs. These data suggest that the down-regulation of LKLF during this critical period of lung morphogenesis may impair lung development and maturation, resulting in pulmonary hypoplasia in the nitrofen CDH model.
肺发育不全是先天性膈疝（CDH）新生儿高发病率和高死亡率的主要原因。然而，与CDH相关的肺发育不全的确切发病机制仍不清楚。最近有报道称，肺Kruppel样因子（LKLF）是转录因子Kruppel样因子家族的成员，主要在肺中表达，在肺形态发生和功能成熟中起重要作用。据报道，小鼠中LKLF基因的纯合缺失导致肺形态发生减少。进一步报道称，源自LKLF（-/-）胚胎干细胞的嵌合小鼠表现出肺发育延迟，尤其是在妊娠后期。因此，我们设计了本研究，以检验以下假设：在硝呋烯腙诱导的发育不全肺中，LKLF基因在肺发育后期被下调。
妊娠大鼠在妊娠第9天（D9）暴露于橄榄油或硝呋烯腙。在D15、D18和D21采集胎儿肺，并分为3组：对照组、无CDH的硝呋烯腙组（CDH（-））和有CDH的硝呋烯腙组（CDH（+））（每组n = 24）。进行实时RT-PCR分析以研究LKLF的肺基因表达水平。对每个时间点的3组之间的差异进行统计学检验，p<0.05时接受显著性。还进行了免疫组织化学以评估LKLF蛋白的表达和分布。
与对照组相比，CDH（-）组和CDH（+）组在D18和D21时LKLF的相对mRNA表达水平显著降低（p<0.01）。硝呋烯腙组和对照组在D15时LKLF的基因表达水平无显著差异。免疫组织化学研究显示，与对照组相比，在D18和D21时硝呋烯腙诱导的发育不全肺中LKLF免疫反应性较强，而在D15时未见差异。
我们的结果首次提供了证据，表明在硝呋烯腙诱导的发育不全肺中，LKLF在肺发育后期被下调。这些数据表明，在肺形态发生的这一关键时期，LKLF的下调可能会损害肺的发育和成熟，导致硝呋烯腙CDH模型中的肺发育不全。
