The Study On Influence Of Transcriptional Profiles Of Porcine Kidney-15 Cell Cultures Following Infection With Porcine Parvovirus

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.