气管插管和口咽吸入两种方法吸入二乙酰构建闭塞性细支气管炎小鼠模型的比较研究

Comparison of establishment of bronchiolitis obliterans murine model by oropharyngeal aspiration and intratracheal instillation of diacetyl

目的比较不同给药方式吸入二乙酰(Diacetyl,DA)对构建闭塞性细支气管炎小鼠模型的差异,探讨简单、易于操作、成功率高且稳定的闭塞性细支气管炎小鼠模型的构建方法。方法将SPF级C57BL/6雄性小鼠(6~8周)按随机数字表法分为4组:口咽吸入组(OPR)、气管插管组(ITI)、口咽吸入对照组(OPR-CON)、气管插管对照组(ITICON),每组10只。OPR组在初次口咽吸入DA(即400mg/kg,327mg/mL)1次,制作闭塞性细支气管炎小鼠模型,ITI组通过气管插管吸入DA(400mg/kg,327mg/mL)1次,OPR-CON和ITI-CON分别经口咽和气管插管,给予等量的蒸馏水代替DA,其余实验条件相同。小鼠饲养于SPF级动物中心,第7天收集标本,分别留取支气管肺泡灌洗液(BALF)和左肺行病理学检测。结果经肺功能、BALF计数和病理切片验证,采用口咽吸入和气管插管一次性给予DA(400mg/kg,327mg/mL)均可成功制备闭塞性细支气管炎小鼠模型,两种方法给予DA后肺功能显示气道阻力增加(P<0.05),肺组织病理切片显示气道上皮损伤,管腔闭塞,支气管周围和血管周围大量炎症细胞浸润等,但OPR组死亡率高达60%,造模成功率仅为20%,肠道HE显示上皮损伤。而ITI组死亡率仅约30%,造模成功率高达60%。OPR-CON组和ITI-CON均未有死亡。结论通过口咽吸入和气管插管两种方法一次性给予DA(400mg/kg,327mg/mL),都能成功构建闭塞性细支气管炎小鼠模型,但ITI组小鼠死亡率低,稳定性好,成功率高,值得实验推荐使用。

Objective To compare the different methods of administration of diacetyl（DA）-established bronchiolitis obliterans（BO）murine model so as to establish a simple,easy-to-operate and stable BO murine model.Methods SPF grade C57 BL/6 male mice（6 to 8 weeks）were randomly divided into four groups with 10 mice in each group：oropharyngeal aspiration group（OPR）,intratracheal instillation group（ITI）,and control groups（OPR-CON and ITI-CON）.OPR group was treated with DA（400 mg/kg,327 mg/kg）by oropharyngeal aspiration;ITI group received DA（400 mg/kg,327 mg/mL）through intratracheal instillation;OPR-CON group and ITI-CON group were treated with sterilized distilled water instead of DA,while the other experimental conditions were the same as those in OPR and ITI groups.The mice were kept in SPF-class animal center for 7 d to collect specimens.Collected bronchoalveolar lavage fluid（BALF）and the left lung were examined pathologically.Results Male C57 BL/6 mice were treated with a single dose of DA（400 mg/kg,327 mg/kg）by OPR or ITI,which could establish BO model.The successful model was evaluated by pulmonary function,BALF counts and pathological examination.Airway hyperresponsiveness occurred with the two-method resulted BO.And two methods of instilling DA resulted in airway injury,lumen occlusion,infiltration of inflammatory cells in the airway and around the vessels.The mortality rate of mice was up to 60% and the success of model construction was only 20% in BO model by oropharyngeal aspiration of DA,while that in ITI group mortality was only 30%,the success of model construction was up to 60%.There was no death in control groups.Conclusion BO murine model could be successfully established by OPR or ITI of DA（400 mg/kg,327 mg/mL）.However,the BO model was established well by ITI of DA with lower mortality rate.Therefore,ITI of DA-established BO murine model is recommended for use.