Chinoy M R, Volpe M V, Cilley R E, Zgleszewski S E, Vosatka R J, Martin A, Nielsen H C, Krummel T M
Department of Surgery, Hershey Medical Center, Pennsylvania State University 17033, USA.
Am J Physiol. 1998 Apr;274(4):L610-20. doi: 10.1152/ajplung.1998.274.4.L610.
Studies on lung morphogenesis have indicated a role of homeobox (Hox) genes in the regulation of lung development. In the present study, we attempted to modulate the synthesis of Hoxb5 protein in cultured murine fetal lungs after mechanical or chemical stimuli. Murine fetuses at gestational day 14 (GD14) were removed from pregnant CD-1 mice, and lungs were excised and cultured for 7 days in BGJb media. The experimental groups were 1) untreated, unligated; 2) tracheal ligation; 3) supplemented media with either epidermal growth factor (EGF; 10 ng/ml), transforming growth factor (TGF)-beta 1 (2 ng/ml), dexamethasone (10 nM), EGF + TGF-beta 1, or EGF + TGF-beta 1 + dexamethasone. After 3 or 7 days, the cultured lungs were compared with in vivo lungs. Immunoblotting signals at 3 days in culture were stronger than those at 7 days. Western blot analyses showed that ligation, EGF, TGF-beta 1, and EGF + TGF-beta 1 downregulated Hoxb5 protein to approximately 20-70% of Hoxb5 protein levels in unligated, untreated cultured lungs. Furthermore, dexamethasone alone or in combination with EGF and TGF-beta 1 downregulated Hoxb5 protein by > 90% (P < 0.05) signal strength, similar to that seen in GD19 or in neonatal lungs. Immunostaining showed that Hoxb5 protein was expressed strongly in the lung mesenchyme at early stages in gestation. However, by GD19 and in neonates, it was present only in specific epithelial cells. A persistent level of Hoxb5 protein in the mesenchyme after EGF or TGF-beta 1 treatments or tracheal ligation was noted. Hoxb5 protein was significantly downregulated by EGF + TGF-beta 1, and it was least in lungs after dexamethasone or EGF + TGF-beta 1 + dexamethasone treatment. The decrease in Hoxb5 protein was significant only in the groups with dexamethasone added to the media. Thus immunostaining results parallel those of immunoblotting. The degree of Hoxb5 downregulation by dexamethasone or EGF + TGF-beta 1 + dexamethasone was similar to that seen in vivo in very late gestation, which correlated to the advancing structural development of the lung.
关于肺形态发生的研究表明，同源框（Hox）基因在肺发育的调控中发挥作用。在本研究中，我们试图在机械或化学刺激后，调节培养的小鼠胎儿肺中Hoxb5蛋白的合成。将妊娠第14天（GD14）的小鼠胎儿从怀孕的CD-1小鼠中取出，切除肺并在BGJb培养基中培养7天。实验组包括：1）未处理、未结扎；2）气管结扎；3）培养基中添加表皮生长因子（EGF；10 ng/ml）、转化生长因子（TGF）-β1（2 ng/ml）、地塞米松（10 nM）、EGF + TGF-β1或EGF + TGF-β1 + 地塞米松。3天或7天后，将培养的肺与体内的肺进行比较。培养3天时的免疫印迹信号强于7天时。蛋白质印迹分析表明，结扎、EGF、TGF-β1以及EGF + TGF-β1可将Hoxb5蛋白下调至未结扎、未处理的培养肺中Hoxb5蛋白水平的约20%-70%。此外，单独使用地塞米松或与EGF和TGF-β1联合使用时，Hoxb5蛋白下调> 90%（P < 0.05），信号强度与GD19或新生肺中的相似。免疫染色显示，Hoxb5蛋白在妊娠早期的肺间充质中强烈表达。然而，到GD19和新生儿期，它仅存在于特定的上皮细胞中。在EGF或TGF-β1处理或气管结扎后，间充质中Hoxb5蛋白水平持续存在。EGF + TGF-β1可显著下调Hoxb5蛋白，在地塞米松或EGF + TGF-β1 + 地塞米松处理后的肺中含量最少。仅在培养基中添加地塞米松的组中，Hoxb5蛋白的减少具有统计学意义。因此，免疫染色结果与免疫印迹结果一致。地塞米松或EGF + TGF-β1 + 地塞米松对Hoxb5的下调程度与妊娠晚期体内观察到的相似，这与肺结构发育的进展相关。
