克隆测序鉴定流感病毒诱导小鼠急性肺炎模型

Identification of mouse acute pneumonia model induced by influenza virus using gene clone and sequence analysis

目的结合RT-PCR病毒载量检测、克隆测序基因比对以及病理改变鉴定甲型流感病毒(influenzavirus,IV)鼠肺适应株(FM1株)诱导的小鼠急性肺炎模型,为急性肺炎模型提供科学的鉴定方案。方法以FM1株滴鼻诱导昆明小鼠急性肺炎,分别于第3、5、7天取小鼠肺脏,以RT-PCR法检测IV病毒载量,并通过质粒克隆测定cDNA序列;同时观察小鼠肺脏病理改变。结果正常组小鼠肺组织的肺泡、肺泡囊和肺泡隔形态完整,周围血管未见炎症细胞浸润;模型组在感染IV后第3、5、7天均见肺泡、肺泡囊、肺泡管、肺泡隔等结构破坏,肺泡间隔增厚,细支气管壁增厚,大量炎症细胞浸润,肺泡壁毛细血管扩张,可见大量红细胞。与正常组比较,模型组在不同时间点的病理变化程度均有显著性差异(P<0.01)。不同时间点模型组小鼠肺组织中均可检测到IV病毒核酸,且不同时间点小鼠肺组织的病变程度与病毒载量呈正相关(P<0.01)。经测序比对发现,IV感染小鼠肺组织中RT-PCR扩增产物与已知IV cDNA序列相应片段吻合度高达99.1%。结论克隆测序基因比对法可鉴定FM1株诱导的小鼠急性肺炎模型。

Objective To identify mouse acute pneumonia model induced by influenza virus adapted strains （FM1 strain） using RT-PCR, gene clone and sequence analysis and pathological examination of lung tissues. Methods Acute pneumonia was induced by intranasal drip of FM1 strain. The lungs were collected after 3, 5 and 7 days. RT-PCR was used to detect the viral load. Amplified PCR products were cloned and sequenced. Pathological and histological changes to the lungs were observed. Results There were no abnormalities in the alveoli, alveolar sacs and alveolar septa and no inflammatory cell infiltration was found in normal mice. In the model group, we found disappearance of alveoli, alveolar sacs, alveolar ducts and alveolar septa, thickening of the alveolar septal and bronchiolar walls, and infiltration of inflammatory cells after 3, 5 and 7 days of influenza virus （IV） infection. Compared with the normal group, pathological changes at various time points were significantly increased （ P 〈 0.01 ）. Viral nucleic acid can be detected in the lung tissue of the model group at various time points, and the pathological changes of the lung tissue were positively correlated with viral load. Sequence analysis demonstrated that there was 99. 1% consistency between RT-PCR products of lung tissues in the model group and the known IV eDNA sequence （ P 〈 0.01 ）. Conclusions Gene clone and sequence analysis may be used to identify acute mouse pneumonia model induced by FM1 strain.