| Objectives:To constuct lentivirus expression vector expressing dendritic cell-specific ICAM-3-grabbing non-integrin-related protein(DC-SIGNR),and design two EBV infection modes including direct infection and the indirect infection of epithelial cells through co-culture with lmphoma cells.This study aimed to further investigate whether DC-SIGNR could play a role in epithelials infection of EBV,laying a foundation for clarifying the mechanism of nasopharyngeal epithelial cells infection by EBV.Methods:1.EBV particles preparation: EBV-GFP was obtained from astric adenocarcinoma cell AGS-EBV-GFP;2.KMH2 cells were directly cultured with EBV,and observed with fluorescence microscope;3.mode of direct contact infection of candidate NPC cells and nasopharyngeal epithelial cells by EBV:each cell line,including HONE1,HK1,CNE1 and NP69 was mixed with prepared EBV,and subsequently the EBV-related molecules,such as m RNA of EBERs,EBNA1,LMP1 and LMP2 A were detected using RTFQ-PCR;4.HONE1 cells and normal immortalized nasopharyngeal epithelial cells were transfected by lentivirus vector to upregulate their DC-SIGNR expressions,following by direct infection by EBV particles.And RTFQ-PCR was used to detect EBV-related molecules,m RNA of EBERs,EBNA1,LMP1 and LMP2 A levels,aiming to evaluate the effect of DC-SIGNR on EBV infection;5.DC-SIGNR overexpressing cell model(RAJI-DC-SIGNR)was constructed by transfecting RAJI cell line by lentivirus vector,with a control of RAJI-CONTROL being built by transfecting RAJI with an empty lentivirus vector without DC-SIGNR.Indirect infections of NPC cells and normal nasopharyngeal epithelial cells were performed by co-culturing RAJI-DC-SIGNR or RAJI-CONTROL cells for 48 h.Subsequently,RAJI-DC-SIGNR cells were eliminated using antibody activation of complement-dependent cytotoxicity test,and RTFQ PCR and Western Blot were used to detect EBV-reated molecules expression levels.Results:1.EBV particles with infection capability were sucessfully prepared: green fluoresence in KMH2 cells,which did not present fluoresence previously,was detected after contact with EBV using fluoresence microscope.2.Target fragment of DC-SIGNR in the cells transfected by lentivirus vectors was detected by the genetical sequencing data,indicating sucessful construction of DC-SIGNR expressing lentivirus vectors.3.Green fluoresence in NPC cells and normal immortalized nasopharyngeal cells(NP69),which was in cultured with EBV particles,was not detected before and after DC-SIGNR upregulation.Meanwhile,no significant upregulations of EBERs,EBNA1,LMP2 A and LMP1 were detected according to our RTFQ PCR data.4.Compared to RAJI-CONTROL,upregulation of DC-SIGNR in RAJI-DC-SIGNR cells could significantly promote EBV related molecules expressions in the NPC cells,but not in NP69 cells.(1)the relative expression levels(2-??Ct)of the EBV-related molecules in HONE1 cells co-cultured with RAJI-DC-SIGNR: levels of EBERs(3.42±0.11)and EBNA1(3.21 ± 0.84)m RNA were significantly higher than those of EBERs(1.24 ± 0.27)and EBNA1(1.24 ± 0.27)in HONE1 co-cultured with RAJI-CONTROL cells,with P values being 0.002 and 0.0432,respectively.However,the relative expression levels of LMP1(1.05 ± 0.30vs1.37 ± 0.52)and LMP2A(1.20 ± 0.11 vs1.12 ± 0.12)were not significantly different between the two groups,with P values being 0.4119 and 0.4588,respectively.(2)the relative expression levels(2-??Ct)of the EBV-related molecules in HK1 cells co-cultured with RAJI-DC-SIGNR: levels of EBERs(75.46±10.43),EBNA1(1.45±0.18),LMP1(7.38±3.58)and LMP2A(5.68±1.67)m RNA were significantly higher than those of EBER(1.52±0.46)?EBNA1(1.05±0.05)?LMP1(1.20±0.22)?LMP2A(1.09±0.08)in HK1 co-cultured with RAJI-CONTROL cells,with P values being 0.003,0.0203,0.0407 and 0.009,respectively.(3)the relative expression levels(2-??Ct)of the EBV-related molecules in CNE1 cells co-cultured with RAJI-DC-SIGNR: levels of EBERs(6.82±1.14),EBNA1(4.62±1.77),LMP1(12.27±1.70)m RNA were significantly higher than those of EBER(1.12±0.10),EBNA1(1.46±0.50),LMP1(2.08±0.94)in HK1 co-cultured with RAJI-CONTROL cells,with P values being 0.001,0.0401,0.0008,respectively.However,the level of LMP2 A was not significantly different between the two groups(0.99 ± 0.49 vs 0.67 ± 0.31),with its P of0.3938.5.the data of Western-blot showed that : EBNA1 level of each NPC cells co-cultured with RAJI-DC-SIGNR was significantly higher than those with RAJI-CONTROL.In detail,EBNA1 level(grey scale)of HONE1(0.36±0.05),HK1(0.46 ± 0.01)cocultured with RAJI-DC-SIGNR were significantly higher than those(0.22±0.05,0.41±0.001)co-cultured with DC-CONTROL,with P values being 0.0267,0.002,respectively.However,the two groups were not significantly different for EBNA1 protein level(0.50 ± 0.04 vs 0.45 ± 0.04,P=0.2168).6.RTFQ-PCR data showed no significant differences of EBERs,EBNA1,LMP1 and LMP2A m RNA in normal NP69(immortalized nasopharyngeal epithelial cells)between the groups of pre-and post-upregulations of DC-SIGNR,both in EBV direct infection mode and indirect infection mode co-cultured with RAJI cells.Conclusions:1.In our study in vitro,EBV may not directly infect NPC cells and nasopharyngeal epithelial cells.2.EBV could infect NPC cells through indirect infection mode of co-culture.3.DC-SIGNR may play an important role as an indirect receptor rather than an direct one in NPC cell infection by EBV in indirect infection mode of cell-cell contact.4.In our in vitro study,EBV could not infect the normal nasopharyngeal immortalized cells both in direct infection mode and the cell-cell contact indirect mode of infection. |
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