摘要
猪细小病毒(Porcine Parvovirus, PPV)是引起妊娠母猪繁殖障碍的主要病原体之一,可引起初产母猪产出死胎、畸形胎、木乃伊胎及病弱仔猪等,还可引起感染母猪不孕或反复发情,并普遍存在于国内外的养殖场,严重影响和制约着养猪业的发展。细胞因子(Cytokine,CK)是由免疫细胞和某些非免疫细胞活化后产生的高活性多功能的小分子多肽、糖蛋白或蛋白,具有调节炎症反应和免疫应答、抗肿瘤和抗病原微生物感染等多种生物学功能。为了进一步了解猪细胞因子对PPV感染的免疫反应机制,更深入的阐明PPV的致病机理,进行了以下研究:
1利用实时定量PCR分别对PPV感染PK-15细胞后1 h、2 h、3 h、12 h、24 h、48 h、72 h细胞中的PPV DNA水平以及1 h、2 h、12 h、24 h、48 h、96 h时细胞中的IFN-β、IFN -γ、IFNAR-1、IFNAR-2、MHC-Ⅰ、MHC-Ⅱ、MX1、NOS、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PK-15细胞后1 h即能检测到PPV DNA,12 h后PPV开始迅速增殖,在感染后48 h达到峰值,为1800倍左右。IFN-β、IFN -γ、IFNAR-2、MHC-Ⅰ、iNOS表达量在感染后1 h并未见增加,但在2 h时增加明显,其中IFNAR-2达到未感染时的21倍左右,随后表达水平逐步降低;IFNAR-1在1 h时基因表达量开始增加,在2 h达到高峰,随后表达水平开始下降;MHC-Ⅱ、Mx1表达在感染后12 h显著增加,其中Mx1基因在24 h表达量达到8423倍,此后迅速下降;2-5OAS、RNaseL在48 h达到高峰,其它时间表达差异不显著;IRF-3在感染后1 h表达量显著增加,达到97倍,在2 h和12 h仍增加显著,48 h之后降低。
2利用实时定量PCR分别对PPV感染PK-15细胞后1 h、2 h、12 h、24 h、48 h、96 h的细胞中的TNF-α、TNFR2、Bcl-2、Bcl-xl、FasL、p53、Caspase-8和PBR等凋亡相关细胞因子mRNA分泌水平进行检测,结果发现PPV感染PK-15细胞后TNF-α在2 h表达量明显增加,达到15倍,随着时间的延长表达量降低,在96 h后表达量变化不显著;TNFR2在2 h表达量显著增加,自12 h后表达量降低明显;Bcl-2 mRNA从12 h到48 h表达量明显增加,96 h后变化不显著;Bcl-xl在2 h时表达量显著增加,达到9.6倍,随后逐渐降低;FasL在12 h表达量增加显著,24 h后表达量下降显著;2 h时p53的表达量显著增加,24 h后表达量变化不明显;2 h时Caspase-8表达量显著增加,达到1.97倍,随后逐渐降低;PBR表达量从2 h开始增加,24 h达到41倍之多,随后表达量开始下降。
3利用实时定量PCR分别对PPV感染PK-15后1 h、2 h、12 h、24 h、48 h、96 h的细胞中的IL-1β、2、6、8、10、12p35、12p40、13、15、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PK-15细胞后在1 h时IL-1β、IL-8、IL-12P40的表达量明显增加,在2 h时达到峰值,分别达到33倍、8倍、32倍;IL-6的表达量在1 h显著增加,达到19倍,12 h后变化不显著;IL-12P35表达量1 h时开始增加,12 h达到峰值;IL-13在2 h时表达量增加显著,随后逐渐下降;IL-17在PPV感染后表达量显著下降,甚至低于对照组细胞的表达量;IL-18基因在2 h表达量即显著增加,随后持续稳定高效表达。PK-15细胞不分泌IL-2、IL-10及IL-15。
4利用实时定量PCR分别对PPV感染PK-15后1 h、2 h、12 h、24 h、48 h、96 h细胞中MIP-1α、TGF-β1、PGK1和MURR1分泌水平进行检测,结果发现MIP-1α在感染后1 h表达量即显著增加,在2 h达到峰值,基因表达量差异倍数为13倍;TGF-β1在感染后96 h表达变化量达到8.8倍,而在其余时间点检测结果均无显著变化;PGK1在感染后2 h的表达量即达到95倍之多,随后开始下降,但仍高于对照组细胞的基因表达量;MURR1在感染后2 h即开始大量表达,12 h达到81倍,24 h迅速达到301倍,48 h时仍为对照组细胞的基因表达量的12倍,至96 h无显著变化。
5利用实时定量PCR分别对不同感染复数PPV感染PK-15后1 h、2 h、12 h、24 h、48 h细胞中的IFN-β、IRF-3、TNF-α和IL-18分泌水平,结果发现IFN-β在感染后1 h表达量开始增加,当12 h且MOI=1时表达量增加显著,达到11倍,此后开始下降;IRF-3在感染后1 h表达量显著增加,在1 h且MOI=10时表达量达到967倍;TNF-α在1 h时表达量开始增加,在2 h且MOI=1时表达量的差异倍数达到96,48 h时表达量开始下降;IL-18从1 h到48 h持续表达,在24 h且MOI=1时表达差异倍数达到64倍。
综上所述,本试验分别对PPV的增殖规律,干扰素及相关细胞因子、凋亡相关细胞因子、炎性细胞因子、部分抗病毒基因及不同感染复数的PPV感染后PK-15部分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理及PPV的分子作用机制提供试验依据,为预防和治疗PPV相关药物的筛选及研制奠定了理论基础。
Porcine Parvovirus (PPV) is one of the major pathogens responsible for reproductive failure of pregnant sows. PPV can cause infected primiparous pregnant sows output fetal death, fetal malformation and the mummified, infected sows can cause infertility or repeated estrus. PPV widespread and prevalent in farms at home and abroad. PPV seriously affected and restricted the development of the pig industry. Cytokines (CK) is a highly active multi-functional small molecule peptide, glycoprotein, or protein which was secrete by immune cells and certain non-immune cell activation. It can regulate inflammation and immune response, anti-tumor and microbial infection of the original disease and other biological functions. To better understand the mechanism of porcine cytokines, further clarify the mechanism of PPV, we carried out the following studies:
1. The PPV DNA levels and gene expression levels of transcripts from 11 cellular genes of PK-15 cell cultures infected with porcine parvovirus after 1 h, 2 h, 3 h, 12 h, 24 h, 48 h, 72 h and 1 h, 2 h, 12 h, 24 h,48 h, 96 h were detected by quantitative real-time PCR SYBR greenⅠm ethod. Of the PPV DNA levels tested, an hour after infection the virus DNA can be detected, 48 h after infection, the peak value was 1800 times. After infected, IFN-β, IFN-γ, IFNAR-2, MHC-Ⅰ, iNOS expression increased significantly in 2 hours, especially the expression levels of IFNAR-2 increased about 21 times; IFNAR-1 reached a peak in 2 h,then the expression began to decline; MHC-Ⅱ, Mx1 expression was increased significantly after infection in 12 h, which Mx1 gene in the 24 h expression level reached to 8423 times, then the expression was gradually decreased; 2-5OAS and RNaseL in 48 h reached a peak expression but at other times was not significant; the expression of IRF-3 was significantly increased after infection 1 h, reaching 97 times at 2 h and 12 h still increased significantly, 48 h after the expression was reduction.
2. The gene expression levels of transcripts from 8 cellular genes of PK-15 cell cultures infected with porcine parvovirus after 1 h, 2 h, 12 h, 24 h, 48 h, 96 h were detected by quantitative real-time PCR SYBR greenⅠmethod. Of the genes levels tested, TNF-αexpression was significantly increased in the 2 h, reaching 15 times, with the time expression decreased, after 48 h the expression did not change significantly; TNFR2 in 2 h expression increased significantly, however, the expression decreased markedly in 12 h; from 12 h to 48 h, Bcl-2 mRNA expression was significantly increased, 96 hours after infection, the change was not significant; in the 2 h, the expression of Bcl-xl increased significantly, reaching 9.6 times, and then declined; in the 12 h FasL expression was increased significantly, but after 24 h, the expression was significantly decreased; in 2 h, the P53 expression was significantly increased, but after 48 h, the expression did not change significantly; the Caspase-8 expression increased significantly in 2 hours, reaching 1.97 times, and then declined; PBR expression began to increase from 2 h, in24 h reach to 41 times, then started to decline.
3. The gene expression levels of transcripts from 11 cellular genes of PK-15 cell cultures infected with porcine parvovirus after 1 h, 2 h, 12 h, 24 h, 48 h, 96 h were detected by use of the quantitative real-time PCR SYBR greenⅠmethod. Of the genes levels tested,an hour after PK-15 cells infected with PPV, the IL-1β, IL-8, IL-12P40 expression was significantly increased, and reached the peak at 2 h, the value is 33 times, 8 times, 32 times; the expression of IL-6 increased significantly in 1 h, reaching 19 times, after 12 hours, the expression was not significant; the expression of IL-12P35 reached the peak at 12 h; IL-13 expression was 1.5-fold in 2 h, and then began to decrease;after PPV infected, IL-17 expression significantly decreased, even lower than the expression of the control cells; IL-18 expression is significantly increased in 2 h, followed by sustained high expression; PK-15 cells do not secrete IL-2, IL-10 and IL-15.
4. The gene expression levels of transcripts from MIP-1α, TGF-β1, PGK1 and MURR1 of PK-15 cell cultures infected with porcine parvovirus after 1 h, 2 h, 12 h, 24 h, 48 h, 96 h were detected by use of the quantitative real-time PCR SYBR greenⅠmethod. Of the genes levels tested, after infection 1 h, the expression of MIP-1αis significantly increased, in the 2 h, the peak value reached 13 times; the expression of TGF-β1 reached 8.8 times in infected 96 h, while in the remaining time the test results did not change significantly; 2 hours after infection,PGK1 expression is 95 times, then decreased, but still higher than the control cell gene expression; 2 hours after infection,MURR1 expressed in large quantities, in 12 h, the value up to 81 times, the value is 301 times, the expression is no significant changes in 96 h.
5. The gene expression levels of transcripts from 4 cellular genes of PK-15 cell cultures infected with three multiplicity of infection after 1 h, 2 h, 12 h, 24 h, 48 h, 96 h were detected by use of the quantitative real-time PCR SYBR greenⅠmethod. Of the genes levels tested, after infection 1 h, the expressions of IFN-βbegan to increase, when in the 12 h and MOI = 1, expressed increased significantly, reaching 11 times, then began to decline; the expression of IRF-3 was significantly increased from 1 h to 48 h, which in the 1 h and MOI = 10, expressed reached the peak, the value is about 967 folds; TNF-αexpression in large quantities when in 1 h, in 2 h and MOI = 1, the differences in the expression of multiple is the largest; from 1 h to 48 h ,IL-18 Sustained high expression continuous, in 24 h and MOI = 1, Peak reached 64 times.
In summary, the expression of PPV DNA, interferon and related cytokines, apoptosis-related cytokines, inflammatory cytokines, some anti-virus gene and some cell cytokines of PK-15 cell after infected by different virus titer of 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.
引文
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