The Study On Human Tonsillar Crypt Epithelial Cells Infected By EV71

Enterovirus 71(Enterovirus 71,EV71)is the most common pathogenic virus causing hand foot and mouth disease(HFMD)of preschool children,especially under 5 years old.It can cause serious central nervous system disease,and its pathogenesis is still unclear.EV71 is mainly transmitted through the fecal-oral pathway,and the oral cavity is the initial site of the virus entering the human body.But the target organ or target cell of EV71 infection is still unclear.Recently,there is a study on the autopsy specimens of children died of EV71 infection,it reported that EV71 antigen and RNA were detected in the tonsillar crypt epithelial,but not in other parts of the gastrointestinal tract.We speculate that the tonsillar crypt epithelial cells may be the target cells that support EV71 replication.However,there are few studies and reports about the EV71 infection of human tonsillar crypt epithelial cells and the immune response of human tonsillar crypt epithelial cells to EV71 is little known.Therefore,this study is divided into three parts:Part I: Isolation,primary culture and identification of human tonsillar crypt epithelial cells Objective:To establish stable and efficient method for primary culture of human tonsillar crypt epithelial cells.Methods:Eighty palatine tonsil tissues were derived from children aged from 3 to 5 undergoing surgical removal of tonsil.Tissue piece method and combined type II dispase and 0.25% trypsin-EDTA digestion method were used to separate primary tonsillar crypt epithelial cells and the effectiveness oftwo isolation methods were compared.Serum-free Keratinocyte medium were applied to purification and primary cultivation.Growth feature and morphology of primary cell was observed under an inverted microscope.Immunofluorescence and immunocytochemistry technique were used to identify the specification of primary cell.Results : The success rate,density and primary cell growth cycle of dispase digestion group were all higher than those of tissue piece group(P < 0.05).The primary tonsillar crypt epithelial cell began adherent growth after 2 days isolation from the tonsil tissue.The shape of cell was polygon under microscope,and cells connected like islands.Primary cells formed confluent monolayers after 12 days' cultivation,it seemed like paving stone.Pancytokeratin positive staining was determined by immunocytochemistry.The specific cytokeratin8/18 staining were positive after immunofluorescence identification.Conclusion:It was an efficient and repeatable method for primary isolation and cultivation of human tonsillar crypt epithelial cells by using combined type II dispase and 0.25% trypsin-EDTA digestion method and serum-free keratinocyte medium.Part II: The establishment of cell model of EV71 infection on human tonsillar crypt epithelial cells.Objective: Establishment of vitro model of EV71 infection on human tonsillar crypt epithelial cells.Methods: EV71 was used to infect human tonsillar crypt epithelial cells.The normal control group and EV71 infection group(12h,24 h,36h,48h)were set up respectively.The cell pathological changes of different groups were observed under the microscope,and EV71 nucleic acid fragments were amplified by Real-time PCR.The expression of EV71 protein was detected by Western Blot,and the expression of EV71 virus in human tonsillar crypt epithelial cells were detected by immunofluorescence.Results: The tonsillar crypt epithelial cells have obviouscytopathic effect after EV71 infection.With the prolongation of infection time,the expression of EV71 virus nucleic acid and protein is increasing,and mainly located in the cytoplasm.The new virus particles are released into the supernatant.Conclusion:We first proved that the human tonsillar crypt epithelial cells support the replication and proliferation of EV71,which is a sensitive target cell of EV71,and successfully established cell model of human tonsillar crypt epithelial cells infected by EV71.Part III: Host cell immune response induced by human tonsillar crypt epithelial cells infected with EV71.Objective: To investigate host cell immune response induced by human tonsillar crypt epithelial cells infected with EV71.Methods: Human tonsillar crypt epithelial cells were infected by EV71 and these cells were divided into EV71 infection group and normal control group.The cell RNA was collected at different time point of each group by Tirzol,and the concentration,purity and integrity of RNA were tested.After the quality examination was qualified,the cDNA library was constructed and sequenced by RNA-Sequencing.The results of EV71 infection group of the same time point were compared with that of the normal control group.The differentially expressed genes were enriched and analyzed by the function of KEGG Pathway.The immune related differentially expressed genes at different time points(12h,24 h and36h)were detected in human tonsillar crypt epithelial cells infected by EV71.Results:The RNA-sequencing results showed that the expression of TLRs pathway(MyD88,TRIF,TRAF6,etc.),RLRs pathway(RIG-I,MDA5,etc.),NLRs pathway(NLRP1,etc)in EV71 infection group were up regulated,and the expression of C3 and CD59 in the complement system were significantly increased.The expression of various chemokines(CXCL8,CXCL10 and so on)were up-regulated,and the expression of apoptosis factors(Caspase,APAF1,FLIP,etc.)and anti-apoptotic factors(BIRC3,etc.)were both increased in EV71 infected human tonsillar crypt epithelial cells.A large number of antiviral factors(IFITMs,OAS1,MXs,ISGs,etc.)were up regulated.Conclusion: EV71 infected human tonsillar crypt epithelial cells can activate a strong immune response through various pattern recognition receptors,secreting a large number of cytokines and antiviral factors,and there is a phenomenon of apoptosis and anti apoptosis in EV71 infected human tonsillar crypt epithelial cells.