C/EBP AND ALVEOLAR TYPE II CELL DIFFERENTIATION

C/EBP 和肺泡 II 型细胞分化

• 批准号: 2029281
• 负责人: SHELDON I FEINSTEIN
• 金额: $ 22.03万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-12-01 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2029281
• 关键词: DNA footprinting; antisense nucleic acid; cell differentiation; cell line; developmental genetics; gene expression; laboratory rat; lung alveolus; pulmonary surfactants; respiratory epithelium; transcription factor; transfection; transfection /expression vector

The type II cells of the alveolar epithelium are involved in the synthesis of pulmonary surfactant, a complex of lipids and proteins which lowers surface tension preventing alveolar collapse. Disorders of surfactant are postulated to cause respiratory distress syndrome in premature infants, congenital alveolar proteinosis in newborns and respiratory distress syndrome in adults. The hypothesis underlying this proposal is that the C/EBP family of transcription factors, known to play an important role in cells exhibiting specialized lipid metabolism, is involved in maintaining the differentiated state of the type II cell. C/EBPs can stimulate gene expression in a variety of epithelial cells. C/EBPs were first purified from liver; however, they are found in lungs as well. Preliminary data indicates that C/EBPs alpha, beta and delta are all present in adult rat type II cells. We have shown that when the type II cells are removed from the lung and purified, the level of C/EBPalpha rapidly declines. We have shown that levels of C/EBPalpha mRNA and protein increase late in fetal rat development while levels of C/EBPdelta increase in human fetal lung explants as lung maturation occurs. C/EBPalpha is detectable by immunoslot blotting in NC-H441, a lung-derived cell line which expresses SP-A and SP-B, while transfection of NC-H441 cells with antisense against C/EBPalpha reduces SP-A and SP-B gene expression. Introduction of C/EBPalpha expression vector into A549 cells, which are thought to be derived from type II cells but no longer express surfactant protein genes induces surfactant protein gene expression in these cells. Our hypothesis that C/EBPs play a role in the maintenance of the surfactant system will be tested by: 1) blocking c/EBP expression with antisense RNA or antisense adenoviral vectors in a cell line which produces surfactant proteins A and B as well as in rate type II cells; 2) using adenoviral vectors to induce synthesis of C/EBPs into type II-derived cell lines which no longer express surfactant proteins in order to restore their differentiated properties and also selecting stably transfected sublines which express C/EBPs and 3) examining the ability of C/EBPs to directly bid to sites in the rat SP-A gene and activate SP-A in transfection experiments.

肺泡上皮的II型细胞参与了 脂质和蛋白质复合物肺表面活性物质合成 其降低表面张力，防止肺泡塌陷。 障碍 表面活性剂被假定在以下情况下引起呼吸窘迫综合征 早产儿、新生儿先天性肺泡蛋白沉积症， 成人呼吸窘迫综合征 这背后的假设是 一种建议是，已知的C/EBP家族的转录因子， 在表现出特殊脂质代谢的细胞中起重要作用， 参与维持II型细胞的分化状态。 C/EBP可以刺激多种上皮细胞中的基因表达。 C/EBP首先从肝脏中纯化;然而，它们在肺中被发现。 也 初步数据表明，C/EBP α，β和δ 都存在于成年大鼠的II型细胞中。 我们已经证明，当 从肺中取出II型细胞并纯化， C/EBPalpha迅速下降。 我们已经证明，C/EBPalpha的水平 mRNA和蛋白质在胎鼠发育后期增加， 随着肺成熟，人胎肺外植体中的C/EBP δ增加 发生。C/EBPalpha可通过免疫印迹法在NC-H441中检测到，a 表达SP-A和SP-B的肺源性细胞系，同时转染 NC-H441细胞中的SP-A和SP-B在反义C/EBPalpha的作用下降低 基因表达。 将C/EBPalpha表达载体导入A549中 细胞，这被认为是来自II型细胞，但不再 表达表面活性蛋白基因诱导表面活性蛋白基因 在这些细胞中表达。 我们假设C/EBP在 表面活性剂体系的维持将通过以下测试：1）阻塞 反义RNA或反义腺病毒载体表达c/EBP， 细胞系，其产生表面活性剂蛋白A和B，以及在速率 II型细胞; 2）使用腺病毒载体诱导C/EBP的合成 转化为不再表达表面活性剂的II型衍生细胞系 蛋白质，以恢复其分化特性， 选择表达C/EBP的稳定转染的亚系和3） 检查C/EBP直接投标到大鼠SP-A中的位点的能力 基因并激活SP-A。