A primary culture model of differentiated murine tracheal epithelium
A primary culture model of differentiated murine tracheal epithelium
复制标题
DOI：
10.1152/ajplung.2000.279.4.l766
复制
发表时间：
2000-10-01
影响因子：
4.9
通讯作者：
Dorin, JR
中科院分区：
文献类型：
--
作者：
Davidson, DJ;Kilanowski, FM;Dorin, JR
本研究的目的是建立一种分化的小鼠气管上皮的原代培养模型。当在空气界面的半透膜上生长时，分离的小鼠气管上皮细胞形成融合的极化上皮细胞，具有高的跨上皮阻力(类似于12k Omea.cm(2))，可存活长达80天。免疫组织化学及光镜和电子显微镜观察显示，细胞为上皮样细胞(细胞角蛋白阳性，波形蛋白和α-平滑肌肌动蛋白阴性)，从接种后第8天开始分化为纤毛细胞和分泌性细胞。用RT-PCR检测囊性纤维化跨膜传导调节因子(CFTR)和小鼠β-防御素(Defb)基因的表达(Defb-1是结构性表达，而Defb-2是内毒素诱导的)。最后，Ussing小室实验证明了一种与功能性阿米洛利敏感的钠通道和cAMP刺激的CFTR氯通道兼容的电生理特性。这些结果表明，原代培养的小鼠气管上皮具有许多与体内小鼠气管上皮相似的特性。该方法将有助于从人类疾病转基因小鼠模型中建立原代培养的呼吸道上皮细胞。
The goal of this study was to develop a primary culture model of differentiated murine tracheal epithelium. When grown on semipermeable membranes at an air interface, dissociated murine tracheal epithelial cells formed confluent polarized epithelia with high transepithelial resistances (similar to 12 k Omega.cm(2)) that remained viable for up to 80 days. Immunohistochemistry and light and electron microscopy demonstrated that the cells were epithelial in nature (cytokeratin positive, vimentin and alpha-smooth muscle actin negative) and differentiated to form ciliated and secretory cells from day 8 after seeding onward. With RT-PCR, expression of the cystic fibrosis transmembrane conductance regulator (Cftr) and murine beta-defensin (Defb) genes was detected (Defb-1 was constitutively expressed, whereas Defb-2 expression was induced by exposure to lipopolysaccharide). Finally, Ussing chamber experiments demonstrated an electrophysiological profile compatible with functional amiloride-sensitive sodium channels and cAMP-stimulated CFTR chloride channels. These data indicate that primary cultures of murine tracheal epithelium have many characteristics similar to those of murine tracheal epithelium in vivo. This method will facilitate the establishment of primary cultures of airway epithelium from transgenic mouse models of human diseases.