The Regulative Action Of IRPS On Porcine MoDC Infected With PRV In Vitro

On the basis of the successful model of dendritic cells derived from porcine peripheral blood mononuclear cells(Mo DC)in vitro,this experiment studies the effect of IRPS on morphology and function of Mo DC,researches interaction of Pseudorabies virus with Mo DC.And we also studied that IRPS regulated the cytokines of Mo DC affected by PRV in vitro.All the results could help us learn the pathogenic mechanism of PRV and the immunomodulation mechanism of IRPS on Mo DC.1 The antivirus activity of IRPS on PRV in vitroIn this experiment,IRPS was extracted from isatis root by water-extraction and ethanol-precipitation and was added into ST cells before or after or together with PRV respectively to evaluate its effect on the virus infection through MTT assay.The results showed that: the maximal atoxic concentration(TC0)of IRPS on ST cells is 0.625mg/m L.In the safe concentration scope,IRPS has a good blocking、inhibiting and directly killing action to PRV in vitro,and the directly killing action is more obvious.2 IRPS affect the morphology and function of Mo DC in vitroIn the presence of rp GM-CSF(20ng/m L)and rp IL-4(20ng/m L),porcine PBMC were derived Mo DC.On Day 5,Mo DC were induced with LPS(100ng/m L)as positive control,different concentrations of IRPS was added into Mo DC as experiment groups.CCK-8 method was used to detect the cell activity,and the optimal concentration of IRPS for promoting the proliferation of DC was 3.125μg/m L.The Mo DC post treatment with IRPS exhibited more typical dendritic morphology with more long protrusions and more cascading folds,and releasing higher level of IL-12 and low level of IL-6,and higher expression of cytokines of IL-1β、IL-6、IL-10、IL-12p35 m RNAthan untreated Mo DC.Our study suggest that IRPS play marked immunostimulating role on the maturation of porcine Mo DC.3 Interaction of Pseudorabies virus with porcine Mo DCIn this study,porcine Mo DC were exposed to a standard virulent strain(Min A)of PRV in vitro.The results showed that: PRV g D protein were detected in nuclear and cytoplasmic of Mo DC at 24 h post-infection by immunofluorescence.Viral progeny was detected in supernatant and intracellular of infected-Mo DC,but limited replication was discovered in the first 12 h after infection.Immature DC infected with PRV produce higher levels of IL-1β、IL-10 and IL-12p35 m RNA,low level of IL-6 and IFN-γ m RNA,TNF-αm RNA does not change compared to mock-infected immature DC.LPS-treated DC infected with PRV produce higher levels of IL-12p35 m RNA,low level of IL-1β、IL-6、IL-10 and IFN-γ m RNA compared to mock-infected LPS-treated DC.4 The regulative action of IRPS on Mo DC infected with PRV in vitroIn this study,Mo DC were stimulated with LPS or IRPS for 48 h on cultured day 6,and exposure to PRV for 24h、48h 、72h,and harvesting supermatant and cells.PRV viral particles were measured byfluorescence quantitative PCR(FQ-PCR),and IL-6、IL-12 protein level by ELISA kit,and cytokines m RNA expression by FQ-PCR.The results indicated that IRPS were no impacted on viral copy,and inhibited IL-6 releasing but at the same time,IRPS group higher significantly to LPS group,and enhanced IL-12 releasing,and higher expressed IL-12 m RNA,and low expressed IL-10、IFN-γ m RNA.The results indicated that there was a certain relationship between the immune function and the antiviral effect of IRPS and the cytokine induced by DC.