ISOLATION OF LINES OF TYPE II CELLS

II 型细胞系的分离

• 批准号: 3362684
• 负责人: GARY W HUNNINGHAKE
• 金额: $ 20.13万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3362684
• 关键词: Adenoviridae; Retroviridae; cell differentiation; cell growth regulation; clone cells; epithelium; gene expression; laboratory rat; lung alveolus; neoplastic transformation; tissue /cell culture; virus genetics

Type II alveolar epithelial cells play an important role in the structure and function of pulmonary alveoli. These cells are also thought to be the progenitors of type I cells which cover much of the alveolar surface. Numerous methods have been devised to isolate and study these cells in vitro. Although the studies which have utilized these methods have markedly enriched our understanding of these cells, they are limited by the following observations: 1) Type II cells do not proliferate to any extent in vitro, thus, all studies must be performed on primary isolates of cells. This characteristic limits the number of studies that can be performed. 2) Although the rate of differentiation of type II cells to type I cells can be altered, the cells are constantly changing their differentiated state in vitro. These observations suggest that lines of type II cells which do not significantly change their differentiated state in culture would significantly improve our ability to evaluate these cells. Optimally, these cells should grow but not exhibit typical transformed (malignant) characteristics. Recently, Cone et al showed that a variety of rat epithelial cells (lung cells were not studied) could be immortalized using a retrovirus expressing the 12S adenoviral E1a gene product (Mol Cell Biol 8:1036, 1988). The cells grew in culture but did not exhibit transformed (malignant) characteristics. These observations led to the following overall hypotheses which underlie the proposed studies: 1)Lines of rat alveolar epithelial cells can be generated using a retrovirus expressing the 12S adenoviral E1a gene product, as described by Cone et al (12). These cells will maintain a relatively stable differentiated state i culture. 2)Clones of rat alveolar epithelial cells at different stages of their differentiation can be isolated. 3)The differentiated state of the different clones of cells can be altered in culture by exposure to agents known to affect the differentiated state of epithelial cells. To evaluate these hypotheses, we will ask the following specific questions: 1) Can lines of rat alveolar epithelial cells be generated using the retrovirus expressing the 12S adenoviral E1a gene product that was described by Cone et al? 2) Do these cells exhibit relatively normal (nonmalignant) characteristics? 3) Can clones of alveolar epithelial cells at different stages of their differentiation be isolated and maintained in culture? 4) Do the lamellar bodies in the type II cell lines have a lipid composition similar to that of freshly isolated cells? 5) Do the lines of type II cells produce the surfactant apoproteins, SP-A, SP-B, SP-C? 6) Do clones which contain lamellar bodies release surfactant in response to stimulation with beta agonists? 7) Can the differentiated state of the different clones of alveolar epithelial cells be altered by agents that are known to affect the differentiation of other epithelial cells? These studies, and the cell lines, should markedly enhance our understanding of the structure and function of alveolar epithelial cells in the lung.

II型肺泡上皮细胞在结构中起重要作用 肺肺泡的功能。这些细胞也被认为是 I型细胞的祖细胞覆盖肺泡表面的大部分。 已经设计了许多方法来分离和研究这些细胞 体外。尽管使用这些方法的研究明显 丰富了我们对这些细胞的理解，它们受到 以下观察结果：1）II型细胞不会在任何程度上增殖 因此，在体外，必须对细胞的主要分离株进行所有研究。 这种特征限制了可以执行的研究数量。 2） 尽管II型细胞与I型细胞的分化速率可以 改变，细胞在不断改变其差异化状态 体外。这些观察结果表明，II型细胞线不得 显着改变了他们在文化中的差异化状态 显着提高了我们评估这些细胞的能力。最佳，这些 细胞应生长但不表现出典型转化（恶性） 特征。最近，Cone等人表明了各种大鼠 可以使用上皮细胞（未研究肺细胞）使用 表达12S腺病毒E1A基因产物的逆转录病毒（Mol Cell Biol 8：1036，1988）。细胞在培养中生长，但没有表现出变化 （恶性）特征。 这些观察结果导致了以下总体假设 拟议的研究：1）大鼠牙槽上皮细胞的线可以是 使用表达12S腺病毒E1A基因的逆转录病毒生成 产品，如Cone等人（12）所述。这些细胞将保持 相对稳定的差异化状态I文化。 2）大鼠牙槽的克隆 分化的不同阶段的上皮细胞可以是 孤立。 3）不同细胞克隆的分化状态可以 通过暴露于已知影响的药物来改变文化 上皮细胞的分化状态。 为了评估这些假设，我们将提出以下特定问题： 1）可以使用大鼠肺泡上皮细胞的线 表达12S腺病毒E1A基因产物的逆转录病毒是 Cone等人描述？ 2）这些细胞表现出相对正常的 （非恶性）特征？ 3）肺泡上皮细胞的克隆 在不同的阶段，分化并保持 文化？ 4）在II型细胞系中的层状体是否具有脂质 组成类似于新鲜分离的细胞？ 5）行 II型细胞产生表面活性剂载蛋白，SP-A，SP-B，SP-C？ 6）做 包含层状体的克隆响应于 用β激动剂刺激？ 7）可以区分的状态 肺泡上皮细胞的不同克隆会通过 已知会影响其他上皮细胞的分化？ 这些研究以及细胞系应显着增强我们的 了解肺泡上皮细胞的结构和功能 肺。