Previous studies have shown that pulmonary mesenchyme is required to maintain epithelial viability and to support branching morphogenesis and cytodifferentiation. We have examined whether pulmonary mesenchyme can be replaced by a medium containing a combination of soluble factors. Day 13-14 fetal rat distal lung epithelium was enzymatically separated from its mesenchyme, enrobed in EHS tumor matrix, and cultured for 5 d in medium containing concentrated bronchoalveolar lavage, EGF, acidic fibroblast growth factor, cholera toxin, insulin, and FBS (TGM), or in control medium containing only FBS. After 5 d in culture, marked growth and morphological changes occurred in epithelial rudiments cultured in TGM, whereas no changes were seen in controls. [3H]Thymidine incorporation and nuclear labeling indices during the last 24 h of culture confirmed that epithelial rudiments cultured in TGM had significant proliferative capacities. Evaluation of surfactant protein gene expression by Northern analysis, in situ hybridization, and immunocytochemistry demonstrated that distal lung epithelial differentiation progressed in TGM. Ultrastructural analysis demonstrated that fetal distal lung epithelium cultured in TGM contained lamellar bodies and deposited a basal lamina. These results are the first demonstration that sustained proliferation and differentiation of glandular stage distal pulmonary epithelium can proceed in the absence of mesenchyme.
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机译:先前的研究表明,需要肺间充质维持上皮的生存能力并支持分支形态发生和细胞分化。我们已经检查了肺间质是否可以被包含可溶性因子组合的培养基替代。将第13-14天的胎儿大鼠远端肺上皮酶解与其间充质,包裹在EHS肿瘤基质中,并在含有浓缩支气管肺泡灌洗液,EGF,酸性成纤维细胞生长因子,霍乱毒素,胰岛素和FBS(TGM)的培养基中培养5 d )或仅包含FBS的控制媒体中。培养5 d后,在TGM中培养的上皮瘤细胞发生了明显的生长和形态变化,而对照中没有看到变化。 [3 H]胸腺嘧啶核苷掺入和培养的最后24小时内的核标记指数证实,在TGM中培养的上皮细胞具有明显的增殖能力。通过Northern分析,原位杂交和免疫细胞化学评估表面活性剂蛋白基因的表达表明,远端肺上皮细胞分化在TGM中进展。超微结构分析表明,在TGM中培养的胎儿远端肺上皮含有层状体并沉积了基底层。这些结果是第一个证明,在没有间充质的情况下,腺期远端肺上皮细胞可以持续增殖和分化。
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