Abstract：
BACKGROUND: Pulmonary fibrosis is a progressive disease with high incidence and poor prognosis. It is urgent to explore new therapeutic methods for pulmonary fibrosis. As a new treatment method, gene therapy has attracted more and more attention. CCDC59 is a transcriptional coactivator of SP-B and SP-C. Our study mainly aims to explore the effect of overexpression of CCDC59 gene in pulmonary fibrosis of mice.
METHODS: CCDC59 overexpressing lentivirus was constructed and then concentrated. RT-qPCR, Western blotting, and immunofluorescence assays were used to detect the expression of CCDC59, SP-B and SP-C protein in cell line and lung tissues after infected with lentivirus. Immunohistochemical staining and hematoxylin-eosin staining assays were used to assess the degree of fibrosis and ELISA assay was used to detect the concentrations of inflammatory factors, SP-B, and SP-C in bronchoalveolar lavage fluid of mice. Dynamic changes of mice lung function at various time points were assessed by lung function test assay. HIPPO pathway and proliferation capacity of alveolar type II epithelial cells were evaluated by immunofluorescence staining and Western blotting.
RESULTS: Results showed that endotracheal instillation of CCDC59 overexpressed lentivirus significantly alleviated bleomycin-induced inflammation and pulmonary fibrosis in mice. Overexpression of CCDC59 protein in type II alveolar epithelial cells can enhance the expression of SP-B and SP-C. Overexpression of CCDC59 protein significantly protected against pulmonary inflammatory response and improved lung function of mice. Overexpression of CCDC59 protein significantly alleviated the hyperactivation of HIPPO pathway and increased the proliferative capacity of type II alveolar epithelial cells in lung.
CONCLUSIONS: CCDC59 can alleviate inflammation and pulmonary fibrosis in mice by upregulating the expression of SP-B and SP-C in type II alveolar epithelial cells and alleviating the hyperactivation of HIPPO pathway. Our study offers a new potential treatment for pulmonary fibrosis.
METHODS: CCDC59 overexpressing lentivirus was constructed and then concentrated. RT-qPCR, Western blotting, and immunofluorescence assays were used to detect the expression of CCDC59, SP-B and SP-C protein in cell line and lung tissues after infected with lentivirus. Immunohistochemical staining and hematoxylin-eosin staining assays were used to assess the degree of fibrosis and ELISA assay was used to detect the concentrations of inflammatory factors, SP-B, and SP-C in bronchoalveolar lavage fluid of mice. Dynamic changes of mice lung function at various time points were assessed by lung function test assay. HIPPO pathway and proliferation capacity of alveolar type II epithelial cells were evaluated by immunofluorescence staining and Western blotting.
RESULTS: Results showed that endotracheal instillation of CCDC59 overexpressed lentivirus significantly alleviated bleomycin-induced inflammation and pulmonary fibrosis in mice. Overexpression of CCDC59 protein in type II alveolar epithelial cells can enhance the expression of SP-B and SP-C. Overexpression of CCDC59 protein significantly protected against pulmonary inflammatory response and improved lung function of mice. Overexpression of CCDC59 protein significantly alleviated the hyperactivation of HIPPO pathway and increased the proliferative capacity of type II alveolar epithelial cells in lung.
CONCLUSIONS: CCDC59 can alleviate inflammation and pulmonary fibrosis in mice by upregulating the expression of SP-B and SP-C in type II alveolar epithelial cells and alleviating the hyperactivation of HIPPO pathway. Our study offers a new potential treatment for pulmonary fibrosis.
摘要：
背景：肺纤维化是一种进展性疾病，发病率高，预后差。迫切需要探索治疗肺纤维化的新方法。作为一种新的治疗方法,基因治疗受到越来越多的关注。CCDC59是SP-B和SP-C的转录共激活因子。本研究旨在探讨CCDC59基因过表达在小鼠肺纤维化中的作用。
方法:构建过表达CCDC59的慢病毒,然后浓缩。RT-qPCR,西方印迹,采用免疫荧光法检测慢病毒感染后细胞株和肺组织中CCDC59、SP-B和SP-C蛋白的表达。免疫组化染色和苏木精-伊红染色检测纤维化程度,ELISA检测炎症因子浓度,SP-B,小鼠支气管肺泡灌洗液中的SP-C。通过肺功能测试测定法评估小鼠在不同时间点的肺功能的动态变化。通过免疫荧光染色和蛋白质印迹法评估HIPPO途径和肺泡II型上皮细胞的增殖能力。
结果：结果表明，气管内滴注CCDC59过表达的慢病毒可显着减轻博莱霉素诱导的小鼠炎症和肺纤维化。CCDC59蛋白在II型肺泡上皮细胞中的过表达可增强SP-B和SP-C的表达。CCDC59蛋白过表达对小鼠肺部炎症反应有明显的保护作用,改善了小鼠的肺功能。CCDC59蛋白的过表达显著减轻了HIPPO通路的过度激活,增加了II型肺泡上皮细胞的增殖能力。
结论：CCDC59可以通过上调II型肺泡上皮细胞SP-B和SP-C的表达，减轻HIPPO通路的过度活化，从而减轻小鼠炎症和肺纤维化。我们的研究为肺纤维化提供了一种新的潜在治疗方法。
方法:构建过表达CCDC59的慢病毒,然后浓缩。RT-qPCR,西方印迹,采用免疫荧光法检测慢病毒感染后细胞株和肺组织中CCDC59、SP-B和SP-C蛋白的表达。免疫组化染色和苏木精-伊红染色检测纤维化程度,ELISA检测炎症因子浓度,SP-B,小鼠支气管肺泡灌洗液中的SP-C。通过肺功能测试测定法评估小鼠在不同时间点的肺功能的动态变化。通过免疫荧光染色和蛋白质印迹法评估HIPPO途径和肺泡II型上皮细胞的增殖能力。
结果：结果表明，气管内滴注CCDC59过表达的慢病毒可显着减轻博莱霉素诱导的小鼠炎症和肺纤维化。CCDC59蛋白在II型肺泡上皮细胞中的过表达可增强SP-B和SP-C的表达。CCDC59蛋白过表达对小鼠肺部炎症反应有明显的保护作用,改善了小鼠的肺功能。CCDC59蛋白的过表达显著减轻了HIPPO通路的过度激活,增加了II型肺泡上皮细胞的增殖能力。
结论：CCDC59可以通过上调II型肺泡上皮细胞SP-B和SP-C的表达，减轻HIPPO通路的过度活化，从而减轻小鼠炎症和肺纤维化。我们的研究为肺纤维化提供了一种新的潜在治疗方法。
