目的:探讨人副流感病毒Ⅲ型(PIV3)感染豚鼠的咳嗽反射敏感性(CRS)变化和机制,以及神经源性炎症在CRS变化和病毒性咳嗽中的作用.方法:豚鼠50只,随机分成正常对照组以及病毒感染6、12、28和42 d组;通过滴鼻方法接种PIV3.Buxco肺功能仪测定CRS.免疫荧光和FQ-PCR方法检测PIV3抗原和核酸.ELISA法检测肺组织匀浆的P物质(SP)含量;FQ-PCR检测肺组织SP受体NK1、辣椒素受体亚型1(VR1)的mRNA水平;免疫组化方法检测肺组织SP、VR1和蛋白基因产物(PGP-9.5)的表达及半定量分析,并对它们各自与CRS的相关性进行分析.结果:各感染组豚鼠CRS均较对照组明显升高,以42 d组为最高.免疫荧光可见PIV3抗原表达;FQ-PCR显示病毒RNA含量在6 d组最高,并随感染时间逐步降低.各组SP和VR1 mRNA,以及12、28和42 d组NK1 mRNA水平均明显高于对照组.6、12及28 d组SP,28和42 d组VR1及42 d组PGP-9.5蛋白表达均增强.相关分析显示,6和28 d组肺匀浆SP含量,12、28和42 d组NK1 mRNA以及各感染组VR1 mRNA均与CRS呈正相关;6、12和42 d组SP的蛋白表达,12和28 d组VR1及12 d组PGP-9.5蛋白表达亦与CRS呈正相关.结论:神经肽的释放增加提示神经源性炎症的发生.CRS和神经肽的时空变化以及两者的正相关提示神经源性炎症在CRS增高和病毒感染性咳嗽中起重要作用.%AIM : To investigate the changes of cough reflex sensitivity ( CRS ) in guinea pigs of human parainfluenza virus type 3( PIV3 ) infection and the mechanisms, as well as the role of neurogenic inflammation in CRS change and the pathogenesis of viral cough.METHODS: Fifty guinea pigs were randomly divided into normal control group and four virus infection groups of post - infection day ( PID ) 6 , 12,28 and 42.PIV3 was inoculated by intranasal instillation.The Buxco system was used to assess cough reflex sensitivity ( CRS ).PIV3 antigen and nucleic acid were detected by immunofluorescence and fluorescence quantitative PCR ( FQ - PCR ).Substance P ( SP ) content in lung homogenates was assayed by ELISA.NK1 receptor of SP and vanilloid receptor suhtype 1 ( VR1 ) mRNA levels in lung tissue were determined by FQ - PCR.The protein expression of lung SP, VR1 and protein gene product ( PGP - 9.5 ) were examined by immunohistochemistry and semi - quantitative analysis.The correlation hetween neuropeptides and CRS was analysed.RESULTS :The CRS of infected guinea pigs was significantly higher than that in control group, with the highest in PID 42.The viral antigen expression can he seen in hoth PIV3 - infected culture cells and lung tissues.The content of PIV3 RNA peaked in PID 6 and showed a decreasing trend with infection time.The SP and VRI mRNA in all infected groups and NK1 mRNA levels in groups of PID 12.28 and 42 increased significantly.The protein expression of SP in PID 6,12 and 28, VR1 in PID 28 and 42 and PGP - 9.5 in PID 42 enhanced obviously.Correlation analysis showed that the content of SP in PID 6and 28 , NK1 mRNA in PID 12, 28 and 42 and VR1 mRNA in all infected groups had a positive correlation with CRS.The protein expression of SP in PID 6,12 and 42, VR1 in PID 12 and PGP9 in PID 12 showed a positive correlation with CRS.CONCLUSION : The increase of neuropeptides releasing in PIV3 - infected groups suggested that neurogenic inflammation was caused.The temporal and spatial variation of CRS and neuropeptides and the positive correlation between both indicated that airway neurogenic inflammation may play a decisive role in the increase of CRS and postinfectious cough caused by viral infection.
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