目的:建立多次气管滴注博来霉素(bleomycin,BLM)诱导的肺纤维化小鼠模型.方法:随机将C57BL/6J小鼠分为空白(control)组(5只)、BLM多次高剂量(BLM-MH)组(10只)、BLM多次中剂量(BLM-MM)组(8只)、BLM多次低剂量(BLM-ML)组(7只)、BLM单次中剂量(BLM-SM)组(6只);采用单次和多次气管滴注BLM的方法制备肺纤维化小鼠模型,并在第56天绘制生存曲线,收集肺组织,计算肺系数,观察苏木素-伊红(hematoxylin-eo-sin,H&E)和Masson染色的肺组织病理变化,检测羟脯氨酸(hydroxyproline,HYP)含量.利用GEO数据库筛选特发性肺纤维化(idiopathic pulmonary fibrosis,IPF)患者的差异表达基因,并对差异基因进行KEGG通路富集分析,以确定与IPF发生相关的通路.通过Western blot检测各组小鼠肺组织中与IPF相关通路的变化.结果:BLM-MH组与BLM-MM组小鼠在第56天的生存率分别为50%和87.5%,其他组无死亡.与control组相比,模型组的肺系数显著升高(P＜0.05或P＜0.01),肺组织损伤、炎症水平和肺纤维化程度显著增加(P＜0.05或P＜0.01),HYP含量呈升高趋势.与BLM-SM组相比,BLM-MH组和BLM-MM组的肺泡炎、纤维化程度和HYP含量均显著升高(P＜0.05或P＜0.01).GEO数据库及京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析结果显示,细胞外基质-受体相互作用通路(extracellular matrix-receptor interaction pathway,ECM-RIP)可能参与IPF的发生.Western blot结果显示,与空白组相比,模型组肺组织中ECM-RIP通路相关蛋白,如磷酸化聚焦粘附激酶(phosphorylated focal adhesion kinase,p-FAK)和磷酸化Src蛋白(phosphorylated Src,p-Src)的表达水平均升高.与BLM-SM组相比,BLM-MH组小鼠肺组织中p-FAK和p-Src蛋白水平均显著升高(P＜0.05).结论:多次气管滴注BLM建立的小鼠肺纤维化模型能够模拟肺纤维化的疾病特征,为IPF机制研究和药物开发提供了有价值的实验模型.
Establishment and evaluation of a model of pulmonary fibrosis induced by multiple administration of bleomycin in mice
AIM:To establish a mouse model of pulmonary fibrosis induced by multiple intratracheal instilla-tions of bleomycin(BLM).METHODS:C57BL/6J mice were randomly divided into five groups:control group(n=5),multiple high-dose BLM(BLM-MH)group(n=10),multiple medium-dose BLM(BLM-MM)group(n=8),multiple low-dose BLM(BLM-ML)group(n=7),and single medium-dose BLM(BLM-SM)group(n=6).The pulmonary fibrosis mod-el was induced by single or multiple intratracheal instillations of BLM.Survival curves were plotted at day 56,and lung tis-sue was collected for lung coefficient calculation.Pathological changes in lung tissue were assessed using hematoxylin-eo-sin(H&E)staining and Masson staining.Indicators of lung fibrosis,such as hydroxyproline(HYP),were measured.Dif-ferentially expressed genes in patients with IPF were screened using the GEO database,and kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis was performed to identify pathways associated with IPF.Western blot was used to detect changes in these pathways in the lung tissues of the model mice.RESULTS:The survival rates were 50%in the BLM-MH group and 87.5%in the BLM-MM group,with no deaths in other groups.Compared with the control group,the BLM-MH and BLM-MM groups showed significantly increased lung coefficients(P＜0.05),lung tissue dam-age,inflammation levels,degree of pulmonary fibrosis(P＜0.05 or P＜0.01),and HYP content.The BLM-MH and BLM-MM groups,compared with the BLM-SM group,showed significantly elevated levels of inflammation,fibrosis,and HYP content(P＜0.05 or P＜0.01).GEO database and KEGG enrichment analysis revealed that the extracellular matrix-recep-tor interaction pathway(ECM-RIP)may be involved in IPF development.The expression levels of ECM-RIP pathway-re-lated proteins,such as phosphorylated focal adhesion kinase(p-FAK)and phosphorylated Src protein(p-Src),were in-creased in the lung tissues of the model mice compared with the control group.Compared with the BLM-SM group,the BLM-MH group exhibited significant increases in the protein levels of p-FAK and p-Src in the lung tissue(P＜0.05).CONCLUSION:The murine model of pulmonary fibrosis established through repeated intratracheal instillations of BLM effectively mimics the pathological characteristics of the disease,providing a valuable experimental model for the investiga-tion of idiopathic pulmonary fibrosis pathogenesis and for the development of therapeutic agents.
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