| BackgroundMalignant lymphoma(ML)is a kind of malignant tumor that derived from the lymph nodes and other lymph hematopoietic tissue,which can be divided into Hodgkin’s lymphoma(HL)and non-Hodgkin’slymphoma(NHL).The incidence and mortality of lymphoma increased year by year.The incidence of NHL is much higher than that of HL,and it is one of the fastest growing malignant tumors,threatening human health seriously.Epstein-Barr Virus(EBV)is the first virus found to be associated with human malignant tumors.It is a human herpesvirus carried by the majority of human population.Epstein-Barr Virus entry into target cells usually results in lytic or latent infection.In the lytic infection.The latent infection is predominantly nonproductive and characterized by expression of six EBV determined nuclear antigens genes(EBNA-1,EBNA-2,EBNA-3A,EBNA-3B,EBNA-3C,and EBNA-LP),three latent membrane proteins genes(LMP-1,-2A,and-2B),BamHIA rightward transcripts,and two abundant EBV-encoded nonpolyadenylated RNAs(EBER1 and EBER2).EBV isolates worldwide can be grouped into type 1 and type 2,a classification based on the EBNA2、EBNA3A、EBNA3B、EBNA3C gene sequence.Type 1 transforms human B cells into lymphoblastoid cell lines much more efficiently than type 2 EBV.Many studies have shown that the neoplastic NK cells possess monoclonal EBV genome,which indicates that latent EBV infection occurs before the clonal expansion.In almost all of the cases of nasal-type NK cell lymphomas,tumor cells contain EBV.This suggests that EBV should play an important role in the pathogenesis of the disease.In EBV-associated NK cell malignancies,there have shown that the gene expression pattern corresponds to type I or II latency.However,the manner of EBV infection has never been elucidated in human NK cells.ObjectiveIdentifie the subtype of NK cell lines infection of EBV;To explore whether EBV can infect NK cells in vitro.Method1.Routine PCR was used to identify infection of EBV type 1 or 2 subtypes in NK cell lines:The DNA of each cell line in the NK cell line preserved in this laboratory was extracted,and design primers according to the the EBNA3c specific coding sequence area,the 1 and 2 subtypes of EBV infection of each NK cell line were identified by routine PCR.2.Epstein-barr virus was extracted from B95-8 cell culture medium and infected with NKL cells.Experimental group:1 x 10~6 cells were taken from the experimental group and centrifuged at 800 rpm for 5min.The lymphocyte was resuspended by0.5ml of EBV concentration.Suspension cells in 37℃incubate for 2 h.Transfer cells into the six-hole plate,add 2 ml RPMI 1640 culture medium containing 10%fetal bovine serum.After 48 hours,the cells were collected for testing.The control group:no EBV concentration,cultured in the same way.3.Routine PCR detection of EBNA-1 and LMP-1 genes:the DNA was extracted from the experimental group and the control group,primers were designed,and results were detected by conventional PCR method.4.The expression of EBNA-1 and LMP-1 proteins was detected by Western blot5.Observe and record the changes of NKL cell before and after EBV infection:NKL cells in the experimental group and control group were observed under the microscope,and the size and shape of the cells were observed.6.Annexin V-PI apoptosis detection:the cells were collected after infection of48h and 72h,and Annexin v-fict was used to detect the apoptosis of NKL cells in each group.7.Statistical methods:SPSS 21.0 software was used for data analysis and drawing.The data were shown as means±standard deviation.Comparison between two samples were conducted by using t-test for homoscedasticity and t’-test for heterogeneity of variance,P<0.05 indicated that the difference is statistically significant.Result1.The YTS,YT,NKYS,SNK and KAI3 cell lines were all EBV type 1infection variants,and NKL and NK92 were EBV negative.2.EBV can cause NKL cells latent infections:extraction of infection after 48hours each cell genome DNA,conventional PCR results showed that the experimental group EBNA1 gene is positive,the control group were negative,and LMP-1 in two groups were negative.Western blot method to detect expression of EBNA-1 protein expression of LMP-1 protein and the results were negative in the two groups.3.EBV cause early phase of apoptosis of NKL cells:the cells gradually show the irregular shape,size is differ,dissolved,cytoplasm burst,after 7 days almost all cell die.After 48 h and 72 h,NKL cell apoptosis rate was(12.16±2.36)%and(45.25±5.42)%(P=0.020,0.001),after infection of 72h,the NKL cell entered early phase of apoptosis.Conclusion1.In NK cell lines,EBV type 1 infection was predominant.2.Epstein barr virus can latent infect NK cells in vitro. |
