克隆测序鉴定流感病毒诱导小鼠急性肺炎模型

谢彬; 王雪峰; 岳志军; 南春红

doi:10.7499/j.issn.1008-8830.2013.02.017

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中国当代儿科杂志 ›› 2013, Vol. 15 ›› Issue (2) : 145-149. DOI: 10.7499/j.issn.1008-8830.2013.02.017

论著·实验研究

克隆测序鉴定流感病毒诱导小鼠急性肺炎模型

谢彬，王雪峰，岳志军，南春红

作者信息 +

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

XIE Bin, WANG Xue-Feng, YUE Zhi-Jun, NAN Chun-Hong

Author information +

文章历史 +

摘要

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（influenza virus, 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。

Abstract

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 cDNA sequence (P<0.01). CONCLUSIONS: Gene clone and sequence analysis may be used to identify acute mouse pneumonia model induced by FM1 strain.

导出引用

谢彬，王雪峰，岳志军，南春红. 克隆测序鉴定流感病毒诱导小鼠急性肺炎模型[J]. 中国当代儿科杂志. 2013, 15(2): 145-149 https://doi.org/10.7499/j.issn.1008-8830.2013.02.017

XIE Bin, WANG Xue-Feng, YUE Zhi-Jun, NAN Chun-Hong. Identification of mouse acute pneumonia model induced by influenza virus using gene clone and sequence analysis[J]. Chinese Journal of Contemporary Pediatrics. 2013, 15(2): 145-149 https://doi.org/10.7499/j.issn.1008-8830.2013.02.017

参考文献

［1］Uyeki T. Antiviral treatment for patients hospitalized with 2009 pandemic influenza A (H1N1)［J］. N Engl J Med, 2009, 361(23): e110.

［2］Baz M, Abed Y, Papenburg J, Bouhy X, Hamelin ME, Boivin G. Emergence of oseltamivir-resistant pandemic H1N1 virus during prophylaxis［J］. N Engl J Med, 2009, 361(23): 2296-2297.

［3］刘友德, 邹志强, 郭砚梅, 李艳芳, 马斌, 李刚. 奥司他韦与改良麻杏石甘汤治疗甲型H1N1流行性感冒的随机对照研究［J］. 中华传染病杂志, 2011, 29(9): 565-569.

［4］吕玉霞, 李娜, 陈钦慧. 麻杏甘石汤加热毒宁注射液治疗儿童疑似甲型H1Nl流感的临床疗效评价［J］. 中医药信息, 2011, 28(2): 51-54.

［5］王玉光, 王晓静, 杜宏波. 6例甲型H1Nl流感确诊病例中西医证治报告［J］. 北京中医药, 2009, 28(6): 403-406.

［6］郭元吉, 程小雯. 流行性感冒病毒及其实验技术［M］. 北京:中国三峡出版社, 1997：91.

［7］张春花, 朱丹, 刘华钢. 流感病毒感染实验动物模型建立的研究进展［J］. 广西医科大学学报, 2011, 28(5): 808-811.

［8］宋康, 骆仙芳, 汪玉冠, 夏永良, 曹羽. 防感煎剂对流感病毒感染小鼠体重以及免疫器官与肺脏的影响［J］. 中华中医药学刊, 2010, 28(2): 239-242.

［9］Centers for Disease Control and Prevention (CDC). Update: influeza activity—United States, 2003-2004 season［J］. MMWR Morb Mortal Wkly Rep, 2003, 52(49): 1197-1202.

［10］Petrosillo N, Di Bella S, Drapeau CM, Grilli E. The novel influenza A (HINI) virus pandemic: An update［J］. Ann Thorac Med, 2009, 4(4): 163-172.

［11］盖建芳, 冀涝, 姚建宏. 甲型H1N1流感重症患儿继发性免疫功能低下［J］. 中国当代儿科杂志, 2010, 12(10): 829-830.

［12］Barnard DL. Animal models for the study of influenza pathogenesis and therapy［J］. Antiviral Res, 2009, 82(2): A110-A122.

［13］Alexander J, Bilsel P, del Guercio MF, Stewart S, Marinkovic-Petrovic A, Southwood S, et al. Universal influenza DNA vaccine encoding conserved CD4+T cell epitopes protects against lethal viral challenge in HLA-DR transgenic mice［J］. Vaccine, 2010, 28: 664-672.

［14］刘崇海, 蒋利萍, 魏钰书. 流感病毒感染动物模型的研究进展［J］. 国际病毒学杂志, 2006, 13(1): 9-12.