人偏肺病毒感染分子流行病学及与哮喘发作的关系

发布时间：2018-05-08 16:55

本文选题：人偏肺病毒 + 呼吸道合胞病毒　；参考：《重庆医科大学》2010年博士论文

【摘要】： PART I:重庆地区儿童以医院为基础的人偏肺病毒感染分子流行病学研究目的:了解重庆地区急性下呼吸道感染(acute lower respiratory tract infection,ALRTI)住院儿童人偏肺病毒(human metapneumovirus, hMPV)分子流行病学特点和临床特征。方法:收集2006年4月到2008年3月重庆医科大学附属儿童医院呼吸科病房因ALRTI住院儿童住院当日的鼻咽吸取物(nasopharyngeal aspirate,NPA)878份,采用荧光定量PCR(real-time PCR)方法检测hMPV基因组RNA,传统PCR方法扩增hMPV阳性标本的F基因进一步确定hMPV感染,扩增G基因用于遗传进化分析。同时采用传统PCR方法检测呼吸道合胞病毒(respiratory syncytial virus,RSV)和冠状病毒NL-63(human coronavirus NL-63,HCoV-NL6),直接免疫荧光法(direct immunofluorescence assay,DFA)检测流感病毒A、B亚型、副流感病毒1、2、3型及腺病毒,明确有无上述常见呼吸道病毒协同感染。结果:采用real-time PCR的方法从878份标本中检测出227份hMPV阳性,阳性率为25.9%。HMPV全年散发,好发季节为冬春季,感染人群主要为2岁以内儿童,尤其是6个月以下小婴儿。HMPV感染流行病学特点和临床特征与RSV相似,与喘息性疾病的发生相关。HMPV可与常见呼吸道病毒协同感染,与RSV协同感染率最高。HMPV与RSV的协同感染可能导致疾病的重症倾向。遗传进化分析显示重庆地区hMPV A和B两个基因型均流行,研究周期内以A2亚型为优势株。结论:HMPV是重庆地区急性下呼吸道感染住院儿童重要的呼吸道病毒病原之一,其感染对6个月以下小婴儿威胁最大。 PART II:哮喘小鼠病毒感染动物模型的建立目的:建立BALB/c哮喘小鼠hMPV/RSV病毒感染的动物模型。方法:哮喘模型判定:清洁级雌性BALB/c小鼠16只,随机分为对照组和OVA组。OVA组在第1d、第14d用OVA致敏,第28-30d用OVA雾化激发,对照组采用PBS致敏激发,第31d检测小鼠气道高反应性(airway hyperreactivity,AHR)、血清中抗OVA-IgE的浓度、支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中IFN-γ和IL-4的浓度、BALF细胞计数和分类计数以及肺组织病理变化。哮喘动物病毒感染模型判定:清洁级雌性BALB/c小鼠48只,随机分为OVA/hMPV组和OVA/RSV组。在第1d、第14d用OVA致敏,第28-37d用OVA雾化激发,第30d雾化后经腹腔麻醉,OVA/hMPV组鼻腔滴入滴度为1O7.0 TCID50/ml hMPV病毒液,OVA/RSV组鼻腔滴入滴度为1O7.0 TCID50/ml RSV病毒液,感染后3d(第33d)、5d(第35d)、7天(第37d)处死小鼠,检测有无hMPV/RSV感染。结果:哮喘模型:OVA组小鼠在雾化过程中出现哮喘急性发作的症状;小鼠肺功能仪检测存在AHR;血清抗OVA-IgE检测水平明显升高;BALF中IFN-γ降低和IL-4升高;BALF中细胞总数明显增多,表现为以淋巴细胞和嗜酸性粒细胞浸润为主的炎症改变。肺组织病理切片表现为支气管细支气管及伴行血管周围大量炎症细胞浸润,肺间质及肺泡腔内可见嗜酸性粒细胞,以上结果均提示哮喘模型建立成功。哮喘动物病毒感染模型:哮喘小鼠感染病毒(hMPV/RSV)后,出现明显体重降低和行为学异常。将感染后3d、5d、7d的小鼠肺组织接种细胞,可以观察发现细胞出现明显细胞病变(cytopathic effects,CPE);肺组织提RNA,作RT-PCR,均能扩增出相应的目的条带;免疫荧光法检测小鼠肺组织接种的Vero-E6细胞/Hep-2细胞发现,细胞胞浆出现绿色荧光,以上结果均提示hMPV/RSV病毒感染动物模型建立成功。结论:成功建立BALB/c哮喘小鼠hMPV/RSV病毒感染的动物模型,有助于后续研究。 PART III:人偏肺病毒、呼吸道合胞病毒感染与哮喘发作的关系目的:了解BALB/c哮喘小鼠感染hMPV/RSV后气道反应性和免疫反应变化情况,初步阐明上述病毒感染与哮喘小鼠气道炎症的关系。方法:清洁级雌性BALB/c小鼠96只,随机分为4组。OVA组:第1d、第14d OVA致敏,第28-37d OVA激发,第30d PBS滴鼻。对照组采用PBS致敏激发。OVA/hMPV组致敏激发同OVA组,第30d滴入滴度为1O7.0 TCID50/ml hMPV病毒液。OVA/RSV组致敏激发同OVA组,第30d滴入滴度为1O7.0 TCID50/ml RSV病毒液。第33d、35d、37d处死小鼠,每次每组8只小鼠。小鼠感染后每日检测体重变化和疾病评分;感染后3d、5d、7d检测小鼠气道反应性、BALF细胞计数和分类计数、BALF中细胞因子水平、肺组织病理改变以及T细胞亚群分布。结果:哮喘小鼠感染hMPV/RSV后,疾病严重度(疾病评分、体重改变、AHR、BALF中细胞数量、病理改变)、炎症因子(IFN-γ和IL-4)、胞内细胞因子(IFN-γ、IL-4、IL-17和Foxp3)均较OVA组改变明显。HMPV感染比RSV感染诱导更弱的IFN-γ反应;早期病理改变更严重。在感染早期,hMPV和RSV感染可以诱导Th17和Treg反应的增强;在感染中后期,hMPV和RSV感染可以诱导Th1和Th2型反应的增强。HMPV感染比RSV感染诱导的Treg反应更强,Th1、Th2和Th17反应更弱。结论:hMPV/RSV病毒感染可以诱导哮喘小鼠哮喘加剧;与RSV感染相比,hMPV感染早期的病理学改变更严重, Treg反应更强,Th1、Th2和Th17反应更弱。
[Abstract]:Study on Molecular Epidemiology of Human Partial Lung Virus Infection Based on Hospital for Children in Chongqing

Objective : To investigate the molecular epidemiological features and clinical characteristics of acute lower respiratory tract infection ( ALRTI ) in hospitalized children with acute lower respiratory tract infection ( ALRTI ) .

Methods : In April 2006 to March 2008 , 878 samples of nasopharyngeal aspirant ( NPA ) were collected from respiratory ward of Affiliated Children ' s Hospital of Chongqing Medical University .

Results : A total of 227 samples were detected from 878 samples by real - time PCR . The positive rate was 25 . 9 % . The positive rate was 25 . 9 % . During the whole year , the positive rate was 25 . 9 % .

Conclusion : Hmpv is one of the most important respiratory viruses in hospitalized children with acute lower respiratory tract infection in Chongqing .

PART II : Establishment of Animal Model of Viral Infection in Asthma Mice

Objective : To establish an animal model for the viral infection in BALB / c asthmatic mice .

Methods : Sixteen healthy female BALB / c mice were randomly divided into control group and OVA group .

Results : The asthma model : OVA group mice had the symptoms of acute asthma attack during the atomization process ; the serum anti - OVA - IgE level was significantly higher than that in BALF ; the number of cells in BALF increased significantly , and the number of cells in BALF increased significantly .

Conclusion : The animal model of BALB / c asthmatic mice is successfully established , which is helpful for the follow - up study .

PART III : THE RELATIONSHIP BETWEEN HUMAN FEVER VIRUS , Respiratory Syncytial Virus Infection and Asthma

Objective : To investigate the changes of airway reactivity and immune response in BALB / c asthmatic mice infected with hPV / RSV , and preliminarily clarify the relationship between the above - mentioned viral infection and airway inflammation in asthmatic mice .

Methods : 96 healthy female BALB / c mice were randomly divided into four groups : OVA group : day 1d , day 14 OVA sensitization , 28 - 37d OVA challenge , day 30d PBS nasal drops . The control group was sensitized by PBS . After infection , the mice were sensitized and challenged daily .

Results : The severity of disease ( disease score , body weight change , AHR , number of cells in BALF , pathological changes ) , inflammatory factor ( IFN - 纬 and IL - 4 ) , intracellular cytokine ( IFN - 纬 , IL - 4 , IL - 17 and Foxp3 ) were significantly higher than those in OVA group .

Conclusion : The results showed that the virus could induce asthma in asthmatic mice . Compared with RSV infection , the pathological changes in early stage were more serious , Treg reaction was stronger , Th1 , Th2 and Th17 responses were weaker .

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R181.3;R562.25

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