| Porcine hemagglutinating encephalomyelitis virus (PHEV) is a member of thegenus coronavirus. PHEV is the pathogenic of porcine hemagglutinatingencephalomyelitis. Acute contagious disease caused by porcine, patients are infectedwith1-3weeks of piglets, and the main features are vomiting, exhaustion, obviousneurological symptoms. The mortality rate is as high as20%-100%. PHEV hastypical neurotropic, delivered by a local infection of peripheral nerves to the centralnervous system, damage to the central nervous system and the typical neurologicalsymptoms. Although the molecular mechanisms of viral pathogenicity unclear, but itwill at least be the immune system plays an important role in the early stage ofinfection of the PHEV. Dendritic cells (dendritic cell, DC) is the strongest knownantigen-presenting cells, but also the initiator of the adaptive immune response. Thevirus can interfere with the function of DC in various ways, and it can evade theidentification of the immune system and even infect DC, making DC become thetransportation and storage sites of pathogeny, resulting in efficient replication andspread of pathogens in the body. Therefore, this experiment studies the pathogenesisof PHEV by studying the interaction relationships of virus and DC. In order to clarifythe role that PHEV infection process of play in DC, this paper carried out the researchby studying the interaction relationships of virus and DC.First, we chose mice as experimental animals, the mice were cultured bonemarrow-derived DC through isolated. Rinse with sterile syringe from mouse bonemarrow cells, the cells40mesh sieve to remove cell debris and impuritiesorganization, Erythrocyte lysate after centrifugation to remove red blood cell and count after washing, by2×105/dish seeded in24-well plates,1ml was added to eachwell containing GM-CSF and IL-4, and10%fetal bovine serum and cultured inRPMI1640nutrient solution, result showed that after cultured DC for7days and10days detected by flow cytometry FITC-CD11c marker positive rate of55%and77%,meet experiment standards.Due to the existence of DC surface markers and phenotypic with DC’s development,maturation, activation of the different stages of change, so we conducted a streamsurface molecule detection, The results showed that PHEV stimulate24h,48hcompared with the control DC, significantly increased the expression of CD40, CD80,molecules; but after inoculation and CD86not been inhibited always low expression.Overall, after PHEV stimulation DC become mature. Also after inoculation on the DC,PCR and immunofluorescence staining for visual observation showed that PHEVexist160bp fragment in the cytoplasm inside DC. The virus can replicate within72hproliferation in cells. DC positive fluorescent signals inside also confirmed this result.Cytokines are secreted by immune cells in the regulation of cell function can besmall peptides. Play an important role on Immune regulation and adaptive immuneresponses, in this experiment we use the ELISA method for detecting IL-10, IL-12,TNF-α, etc. Results of TNF-α, IL-12after the first and then decreased, Compared withthe control group, the concentration of IL-10infected with the extension of PHEVtime gradually increased,72h after the infection reaches3.6times. Since IL-10caninhibit IL-12production, suggesting that IL-10increased resulted in a decrease in theexpression of late IL-12. In addition, IL-10molecules can regulate the expression ofCD40, CD80and other co-stimulation, which is consistent with the results of theperformance changes after PHEV role of DC.Spleen lymphocyte subsets (CD3+CD4+/CD3+CD8+) detection is an importantindicator of the ability of the immune response, increased explain ability to enhancethe immune representatives. After separation of mouse spleen cells stimulated withdifferent time periods PHEV and DC co-cultured72h, by flow of CD3+CD4+/CD3+CD8+were detected. The results showed that after the first decreased and then increased, indicating that the virus stimulated DC did activate T cells. |
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