【作者】 李金磊；

【导师】 魏战勇；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2011， 硕士

【摘要】 猪细小病毒(Porcine Parvovirus, PPV)是引起猪繁殖障碍综合症的主要病原,可引起死产、木乃伊胎、胚胎的早期死亡及不孕不育等。除了临床上导致繁殖障碍,猪细小病毒还能引起皮炎、肠道等疾病。作为一个能显著导致经济损失的疾病,近年来猪细小病毒在世界范围内普遍流行,感染和造成的危害呈上升趋势,给养猪业造成了巨大的经济损失。细胞因子能介导抗病毒状态的建立及募集炎性细胞向感染部位聚集,不仅在抵抗疾病的过程中发挥重要作用,而且是清除感染所必需的。对于猪细小病毒致病机制的研究多集中在病毒的复制及感染过程,而对病毒与细胞间的作用关系、猪细小病毒感染引起宿主细胞因子的变化规律的研究尚未见报道。为了了解猪细胞因子对PPV感染的免疫反应机制,阐明PPV的致病机理,我们进行了以下研究:1.利用实时定量PCR分别对PPV感染猪PBMC后1 h、3 h、6 h、12 h、24 h、48 h、72 h细胞中的PPV DNA水平及IFN-β、IFN-γ、IFNAR-1、IFNAR-2、MHC-Ⅰ、MHC-Ⅱ、MX1、iNOS、2-5OAS、RNaseL和IRF-3等干扰素及相关细胞因子mRNA分泌水平进行检测。结果发现,PPV感染猪外周血淋巴细胞后1 h即能检测到PPV DNA,24 h后PPV开始迅速增殖,在感染后72 h达到峰值,为10倍多。IFNAR-1 mRNA的表达量在1 h、24 h显著增加,其中1 h为89.9倍;IFNAR-2 mRNA的表达量在24 h达到高峰,其他时间无明显差异;IFN-βmRNA的表达量在1 h达到高峰,24 h显著下调;IFN-γmRNA的表达量在48 h显著增加,24 h显著下调;MHC-ⅠmRNA的表达量在1 h、12 h、48 h显著增加,48 h达到高峰;IRF-3、RNaseL mRNA的表达量在48 h显著增加,其它时间表达差异不显著;MX1、2-5OAS mRNA的表达量在24 h、48 h显著增加,24 h达到高峰;MHC-ⅡmRNA的表达量在1 h、3 h、6 h、12 h显著增加,3 h达到高峰;iNOS mRNA在1 h不表达,3 h表达量显著增加,24 h显著下调。PPV感染可引起猪PBMC分泌抗病毒相关因子显著增加。2.利用实时定量PCR分别对PPV感染猪PBMC后1 h、3 h、6 h、12 h、24 h、48 h、72 h细胞中的IL-1β、6、8、12p35、12p40、13、17和18等炎性细胞因子mRNA分泌水平进行检测。结果发现PPV感染猪PBMC后IL-1βmRNA的表达量在1h、3 h、6 h、12 h、24 h显著增加,24 h达到高峰为58倍;IL-6 mRNA的表达量在1h、3 h、6 h、12 h、24 h、72 h显著增加,6 h达到高峰为40.5倍;IL-12p35 72 h显著增加,24 h显著减少;IL-12p40 1 h显著增加,24 h显著减少;IL-8在1 h、3 h、6 h、12 h、24 h、48 h显著增加,24 h达到高峰为59.7倍;IL-13在3 h显著增加,12h、24 h显著减少;IL-17在1 h、48 h显著增加,6 h、12 h、24 h显著减少;IL-18在6 h显著增加。PPV感染可引起猪PBMC分泌炎性相关因子显著增加。3.利用实时定量PCR分别对PPV感染猪PBMC后1 h、3 h、6 h、12 h、24 h、48 h、72 h细胞中的TNF-α、TNFR-2、P53、PBR、Bcl-2、Bcl-xl、Caspase-8和FasL等凋亡细胞因子mRNA分泌水平进行检测。结果发现PPV感染猪PBMC后TNF-αmRNA的表达量在1 h显著增加,随后逐渐降低,24 h显著减少;TNFR-2 24 h显著减少为0.25倍,48 h显著增加为11.5倍;P53在1 h和72 h有轻微增加,6 h、12 h、24 h减少;PBR在1 h、24 h、48 h显著增加,24 h达到高峰为12.9倍;Bcl-2在1 h有轻微增加,其他时间减少,24 h显著减少;Bcl-xl在24 h显著减少,72 h显著增加;Caspase-8在1 h、24 h显著增加,1 h达到高峰为19.3倍;FasL在1 h显著增加,24 h显著减少为0.02倍。PPV感染可引起猪PBMC分泌凋亡相关因子显著变化。综上所述,本试验分别对PPV在猪PBMC中的增殖规律及PPV感染后猪PBMC干扰素及相关细胞因子、炎性细胞因子、凋亡相关细胞因子的转录时相进行了研究,为进一步了解细胞因子的免疫学作用机制及PPV的分子致病机制提供试验依据,为预防和治疗PPV及相关药物的筛选和研制奠定了理论基础。更多还原

【Abstract】 Porcine parvovirus (PPV) is the major causative virus in a syndrome of reproductive failure in swine, which includes stillbirths,mummified fetuses, early embryonic death, and infertility. Moreover, other clinical manifestation besides reproductive failure, such as dermatitis and enteric diseases. Recently, PPV is widespread among swine throughout the world as a signicant economical disease, PPV infection and the harm caused by the upward trend to the pig farming industry and caused great economic losses. as mediate the establishment of an antivirus state and recruit inflammatory cells to the site of infection, Cytokines were clearly not the only factor contributing to disease, And they seemed to be essential for resolution of the infection. The study of pathogenesis of porcine parvovirus were focused on the process of viral replication and infection, while the relationship between cells and virus, variation of cytokines of host cell infected with porcine parvovirus have not been reported. To understand the mechanism of porcine cytokines, clarify the mechanism of PPV, we carried out the following studies:1. The PPV DNA levels and transcript level of cytokines(IFN-β、IFN-γ、IFNAR-1、IFNAR-2、MHC-Ⅰ、MHC-Ⅱ、MX1、iNOS、2-5OAS、RNaseL and IRF-3)of PBMC cultures infected with porcine parvovirus after 1 h, 3 h,6 h, 12 h, 24 h, 48 h, 72 h were detected by quantitative real-time PCR SYBR greenⅠmethod. The virus DNA can be detected an hour after PPV infection, 72 h after infection, the peak value was more then 10 times.The transcript levels of IFNAR-1 were increased significantly on 1 h and 24 h postinoculation(p.i.),especially 1 h p.i the expression levels of IFNAR-1 increased about 89.9 times; IFNAR-2 reached a peak on 24 h p.i, but was not significant at other times;IFN-βreached a peak in 1 h p.i,but decreased on 24 h p.i; The transcript levels of IFN-γwere decreased on 24 h p.i,but significantly increased 48 h p.i; MHC-Ⅰwas increased significantly on 1 h,12 h,48 h,and reached a peak in 48 h; IRF-3、RNaseL were increased significantly on 48 h, but was not significant at other times; MX1、2-5OAS were increased significantly on 24 h, 48 h,and reached a peak in 24 h; MHC-Ⅱwas increased significantly on 1h,3 h,6 h,12 h,and reached a peak in 3 h; PBMC do not secrete iNOS on 1 h p.i,but the transcript levels of iNOS was increased significantly on 3 h p.i, decreased on 24 h p.i. The PPV infection induced PBMC the secretion of anti-virus related cytokines increased significantly.2. The transcript level of cytokines(IL-1β、6、8、12p35、12p40、13、17 and 18)of PBMC cultures infected with porcine parvovirus after 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h were detected by quantitative real-time PCR SYBR greenⅠmethod. Of the genes levels tested, The transcript levels of IL-1βwere increased significantly on 1h,3 h,6 h,12 h,24 h, the peak value was 58 times on 24 h;IL-6 was increased significantly on 1h,3 h,6 h,12 h,24 h,72 h,and reached a peak on 6 h,which was 40.5 times;IL-12p35 and p40 were decreased significantly on 24 h,but increased significantly on 72 h and 1 h respectively;IL-8 was increased significantly on 1h,3 h,6 h,12 h,24 h,48 h,and reached a peak on 24 h which was 59.7 times;IL-13 was increased significantly on 3 h,but decreased significantly on 12 h,24 h;IL-17 was increased significantly on 1 h,48 h,but decreased significantly on 6 h,12 h,24 h;IL-18 was increased significantly on 6 h. The PPV infection induced PBMC the secretion of inflammatory cytokines increased significantly.3. The transcript level of cytokines(TNF-α、TNFR-2、P53、PBR、Bcl-2、Bcl-xl、Caspase-8 and Fasl) of PBMC cultures infected with porcine parvovirus after 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h were detected by quantitative real-time PCR SYBR greenⅠmethod. Of the genes levels tested, The transcript levels of TNF-αwere increased significantly on 1 h, then started to decline, decreased significantly on 24 h; TNFR-2 was decreased significantly on 24 h,which was 0.25 times,but increased significantly on 48 h which was 11.5 times;P53 was increased slightly on 1 h and 72 h,but decreased on 6 h,12 h and 24 h;PBR was increased slightly on 1 h,24 h and 48 h, the peak value was 12.9 times on 24 h;Bcl-2 was increased slightly on 1 h,but decreased other times, decreased significantly on 24 h; Bcl-xl was decreased significantly on 24 h,but increased significantly on 72 h; Caspase-8 was increased significantly on 1h and 24 h, the peak value was 19.3 times on 1 h; Fasl was increased significantly on 1h, but decreased significantly on 24 h, which was about 0.02 times. The PPV infection induced PBMC the secretion of apoptosis-related cytokines changed significantly.In summary, the expression of PPV DNA, interferon and related cytokines, inflammatory cytokines,apoptosis-related cytokines of porcine PBMC after infected with PPV were studied. Provide an experiment support for further understand the role of cytokines in the immune mechanism and the molecular mechanisms of PPV, and establish theoretical foundation for screening and development of prevention and treatment of PPV-related drugs.更多还原