TLR9 Expression And The Immune Mechanism Of Anti-HCV Mediated By TLR9 In Murine Microglia Cells

Objective To analyze the expression of Toll-Like Receptor 9 and cytokines(IL-12,TNF-α,IL-6,MCP-1)after the stimulation of murine microglia (BV2)by hepatitis C virus (HCV),so as to elucidate the immune pathogenesis of HCV in the injury of central nervous system and the antiviral role.Methods BV2 cells were cultured in vitro and divided into 3 groups : HCV-positive serum group, normal serum group (negative control)and blank control group,each group had 20 samples.(1)The supernatant was collected at 24h,the morphology of BV2 cells was observed,meanwhile,the levels of IL-12,TNF-α,IL-6,MCP-1 were detected by ELISA.The changes,including cell morphology and cytokines,provided a reliable basis for the next step.(2) The cells and supernatant were collected at 4,12,20,36h.TLR9 mRNA was detected by RT-PCR and RT-qPCR, TLR9 protein was detected by FCM,and the levels of IL-12,TNF-α,IL-6,MCP-1 were detected by ELISA.Comparison analyse were performed by SPSS13.0 statistical package.Results (1) The inverted microscope showed the morphology of BV2 cells: in vitro ,BV2 growed by semi-adherent condition, cells were elongated or fusiform, cell bodies had slender cell-process and branchs, in the face, there were many small spinous process.But BV2 cells were cultured by DMEM with HCV-positive serum,cells were round,and cell process became coarsen and short, some cells gathered the conglobation.The levels of TNF-α,IL-12,IL-6 in the HCV-positive serum group were higher than those in the other two groups, the differences had statistical significance(FTNF-α=72.627, FIL-12=16.465,FIL-6=19.344,P<0.01); but there were no significant difference between normal serum group and blank control group(P>0.05). (2) RT-PCR and RT-qPCR showed that there was the expression of TLR9 mRNA in BV2 cells.And the level of TLR9 mRNA in HCV-positive serum was higher than that in the other two groups.The difference had statistical significance(FRT-PCR=17.258,P<0.01,FRT-qPCR= 93.257,P<0.01),It was the highest at 4h(P<0.05),but there were no significant difference between normal serum group and blank control group(P>0.05).FCM showed that there was the expression of TLR9 protein in BV2 cells.And the level of TLR9 protein in HCV-positive serum was higher than that in the other two groups.The difference had statistical significance (F=32.762,P<0.01),It was the highest at 20h(F=16.517,P<0.01),but there were no significant difference between normal serum group and blank control group(P>0.05).(3)The levels of MCP-1,TNF-α,IL-12,IL-6 in the HCV-positive serum were higher than those in the other groups(P<0.05),there were no significant difference between normal serum group and blank control group(P>0.05).Conclusions (1)The stimulation of HCV-positive serum make the morphology of BV2 to change,and it may induce the secretions of IL-12,TNF-α,IL-6.The style that BV2 in vitro are stimulated by HCV-positive serum may simulate the immune response mechanism during the infection of CNS by HCV.(2)The expression of TLR9 mRNA and TLR9 protein are elevated after the stimulation of BV2 cells by HCV-positive serum. TLR9 mRNA is highest at 4h and TLR9 protein is highest at 20h, TLR9 may participate in the early inherent immune response during the infection of CNS by HCV,but the difference of expression in time between TLR9 mRNA and TLR9 protein is to be studied further.(3)The stimulation of HCV-positive serum may induce the secretions of MCP-1,TNF-α,IL-12,IL-6.Thus TLR9 may increase the secretions of chemokine,Th1 and Th2 cytokines so as to play a role in immune response during the infection of CNS by HCV.