Zhang L, Zgleszewski S E, Cilley R E, Chinoy M R
Department of Surgery, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey 17033-0850, USA.
J Surg Res. 1998 Feb 15;75(1):66-73. doi: 10.1006/jsre.1997.5259.
To evaluate lung development at the level of gene expression, a comparison was made between normal and hypoplastic murine fetal lungs by using the mRNA differential display technique. We focused on altered gene expressions at gestational day (Gd) 19 in normal and hypoplastic murine lungs. Hypoplastic fetal lungs were created by gavaging pregnant mice at Gd8 with 25 mg of nitrofen (2,4-dichlorophenyl-p-nitrophenyl ether) [1]. Normal as well as gavaged mice were euthanized by an overdose of halothane at Gd19, and fetuses were removed by laparotomy. Lungs were excised and total RNA was extracted from normal and hypoplastic fetal lungs. Differential display technique was carried out using the RNAimage kit (GenHunter Corp., TN). Each reverse transcription and polymerase chain reaction (RT-PCR) was performed using one specific anchor primer H-T11M (5'HindIII-T11A/C/G3') and one arbitrary primer. We have used a total of 3 different anchor primers and 24 arbitrary primers for each sample. There were 20 differentially expressed cDNA clones, either induced, inhibited, enhanced, or reduced in hypoplastic fetal lungs as compared to normal. Of these, one clone (NL2) with reduced expression in Gd19 hypoplastic lungs had 100% homology with mouse nucleosome assembly protein I gene. Another clone (NT5) with induced expression in hypoplastic lungs is an unknown gene. Further, analyses of Northern blots of lungs from various gestational ages showed that the expression of NT5 was induced in hypoplastic lungs at Gd18, whereas in normal lungs it was first expressed at the neonatal stage and was increasingly expressed into adulthood. There is a single hybridized band, approximately 400 bp long for NT5 message. Dexamethasone induced expression of NT5 in normal Gd14 pseudoglandular lungs cultured for 7 days; however, different growth factors did not. Northern blot hybridization of multiple adult mice tissues showed NT5 expression in the lung, intestine, and spleen. The thyromimetic action of nitrofen and the interactive functional pathways of dexamethasone with T3 are known. Therefore, we suggest that the isolation and characterization of NT5 may provide valuable information on the regulation of lung development.
为了在基因表达水平评估肺发育情况，利用mRNA差异显示技术对正常和发育不全的小鼠胎儿肺进行了比较。我们重点关注了妊娠第19天（Gd19）正常和发育不全的小鼠肺中基因表达的变化。发育不全的胎儿肺是通过在妊娠第8天给怀孕小鼠灌胃25毫克硝呋烯腙（2,4 - 二氯苯基 - 对硝基苯醚）[1]来构建的。在Gd19时，对正常小鼠以及灌胃小鼠过量吸入氟烷使其安乐死，然后通过剖腹术取出胎儿。切除肺脏，从正常和发育不全的胎儿肺中提取总RNA。使用RNAimage试剂盒（GenHunter公司，田纳西州）进行差异显示技术。每个逆转录和聚合酶链反应（RT-PCR）使用一种特异性锚定引物H-T11M（5'HindIII-T11A/C/G3'）和一种随机引物。每个样本我们总共使用了3种不同的锚定引物和24种随机引物。与正常胎儿肺相比，在发育不全的胎儿肺中有20个差异表达的cDNA克隆，表现为诱导、抑制、增强或减少。其中，一个在Gd19发育不全的肺中表达降低的克隆（NL2）与小鼠核小体组装蛋白I基因有100%的同源性。另一个在发育不全的肺中表达诱导的克隆（NT5）是一个未知基因。此外，对不同胎龄肺组织的Northern印迹分析表明，NT5的表达在Gd18时在发育不全的肺中被诱导，而在正常肺中它在新生儿期首次表达，并在成年期逐渐增加。NT5的信息有一条约400 bp长的单一杂交带。地塞米松在培养7天的正常Gd14假腺期肺中诱导了NT5的表达；然而，不同的生长因子没有这种作用。对多只成年小鼠组织的Northern印迹杂交显示NT5在肺、肠和脾中表达。硝呋烯腙的甲状腺模拟作用以及地塞米松与T3的相互作用功能途径是已知的。因此，我们认为NT5的分离和鉴定可能为肺发育的调控提供有价值的信息。
