Study Of The Machanism Of Enterovirus71Entrying Into Human Neuroblastoma Cells

ObjectiveStudying the mechanism of Enterovirus71entrying into humanneuroblastoma cells （SK-N-SH) used by different virus endocytosisblockers, thus providing experimental basis for the pathogenesis of EV71infection with nervous system damage，at the same time for development ofnew clinical antiviral therapy and vaccine research.Material and Methods1. VirusEV71was provided by Microbiol Research of Chongqing Center ForDisease Control and Prevention.2.CellsHuman rhabdomyosarcoma cells(RD) were obtained by MicrobiolResearch of Chongqing Center For Disease Control and Prevention,Human neuroblastoma cells(SK-N-SH) were suppied from cell culture ofChildren’s Hospital of Chongqing Medical University. 3.Experimental groupsThis Experiment was divided into three groups:the blank group;Chlorpromazine treatment group (10,15,20μΜ); Nystatin treatment group(10,15,20μg/ml)；Without drugs as the negative control group;Notreatment as the blank group.The SK-N-SH cells were treated withvarious concentrations of inhibitor for one hour and then infected withEV71in serum-free culture medium for1hour at37℃in the presence ofinhibitor. The cells were washed three times with serum-containing culturemedium and after additional24hours of culture in the presence of theinhibitor, the cells were harvested for TaqMan fluorescence quantitativeRT-PCR.4.Experimental Method4.1EV71was propagated in RD cells. The virus titer was quantitatedvia TCID50.4.2MTT assay was conducted to detect the inhibition of SK-N-SHcells used by different virus endocytosis blockers.4.3The EV71mRNA expression was validated by TaqManfluorescent quantitative RT-PCR.Results1. EV71virus were successfully amplified in RD cells, the virus titerwas1×105TCID50.2. With the increase of drug concentrations gradient, the growth rate ofSK-N-SH cell was inhibited. 3. TaqMan fluorescence quantitative RT-PCR showed the SK-N-SHcells were susceptible to Enterovirus71.4. TaqManQPCR showed the expressionof EV71mRNA was inhibitedby pretreatment of chlorpromazine (Chlorpromazine, CPZ)(Ρ <0.05),Nystatin (Nystatin, NT) has little influence (Ρ>0.05).ConclusionWe speculate initially EV71invading into SK-N-SH cells byclathrin-dependent endocytosis.