| Transmissible gastroenteritis (TGE) is an acute highly contagious infectious disease due to transmissible gastroenteritis virus (TGEV) and caused the 7-days-old or less piglets inside a high mortality rate (usually up to 100%). The symptoms of this disease were vomiting, severe diarrhea, dehydration and so on. After the isolation of the virus in China for the first time, there have been reports from the viruses isolated from pig farms all over the country, and the epidemic intensified of TGEV. The disease outbreak primarily in late winter and early spring, but according to surveys in recent years, incidencing this disease also outbreak in late spring and early summer. And the incidence of small and medium sized farms is 15%～23% in Henan province, has caused serious economic losses to the pig industry in China. Now the research focused on the diagnosis, treatment and the direction to the development of vaccine to this disease, but little success was proved. This is mainly because the pathogenic mechanism of TGEV, especially on the molecular mechanism of TGEV was quite understood before.The DC is the largest antigen-presenting cells in bodys. They can directly activate naive T cells, and produce a series of cytokines, and then involve in immune regulation. There had showed that DC played a role in infect and treatment other coronavirus. But there is no reports about TGEV infect in DCThis study on inducing and differentiating pig DC from peripheral blood cells of pigs, to establish a system of inducing and culture of pigs DC in vitro; and study on the infection and the ability of copy of TGEV in DC; and the impact of cytokine secretion of TGEV infect to DC, in order to reveal the interactions between DC and TGEV. We explored and analyzed the infection mechanism of TGEV, in order to enhance the immune effects and provide a theoretical basis to prevent and control TGEV.1. Collected the peripheral blood of healthy pigs and separated the lymph mononuclear cells. Inducing and differentiating the PBMC to dendritic cells combined with GM-CSF, IL-4, and TNF-α.Observed the cell growth and differentiation with the inverted microscope to examine the morphology of immature DC and mature DC. The results showed that:inducing with the combining of rhGM-CSF, rhIL-4 and rhTNF-a, and training for 10 days, we can observed morphological changes in cell culture by inverted microscope, and can see the typical morphology of dendritic cells. The results can confirm that we have successfully built up methodologies that induced and trained pigs’ DC in vitro. This can lay the foundation to further study on the relationship between pigs’DC and animal diseases.2. In order to understand the characteristics and the ability of replicate in DC after TGEV infected. TGEV infected the mature pigs’DC in vitro for 1,3,12,24,48 and 72h. Infected the ST cells with the supernatant, examined the cytopathic effection to determine the titer of the virus. Then extracted RNA and reversed transcription to cDNA from the cells. We amplified the cDNA of TGEV with the methords of PCR and RT-PCR. Analysised whether TGEV can infect the DC as well as the virus can proliferate in DC. The results showed:we can detect the content of virus in DC after TGEV infection 1 h, and with extended of the infection time, the content of TGEV gradually increased in the DC. The result indicated that TGEV can successfully infect, replicate and proliferate in DC.3. In order to understand the impact of cytokines secretion in DC after TGEV infection, we used double antibody sandwich ELISA method to detect the contents of IL-6, IL-10, TNF-a, IFN-a and IFN-y in cell supernatant after TGEV infected DC for 1,3,12,24,48 and 72h. Using Real-time PCR technology to detect the changes of mRNA transcription level of these immune-related factors after TGEV infected mature DC for different times,including IL-6, IL-8, IL-12 p35, and IL-12 p40, IL-17, IL-18. Results showed that expression of protein of IL-6, IL-10 and IFN-a were increased after TGEV infected DC. There is the most significant increased in IFN-α; the content of TNF-a has been decreased at the same time, and there is no significant changes between the content of IFN-y. The DCs after infection TGEV, the the content of IL-12p35 reached secretion peak at 24h, the content of IL-6 and IL-18 reached secretion peak at 48h; as well as the content of IL-12p40 reached peak at 72h. The content of IL-12 p40. and IL-6 and IL-18 rosed slowly, the secretion of IL-8 gradually reduced, and reached lowest until 72h. There is no obviously changes between the secretion volume of IL-17. These results indicate that the DC after TGEV infection can cause changes the expression of cytokines, laid the foundation to further study of the interactions between DC and TGEV. |
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