| Objective:To explore the molecular mechanism of oncolytic reovirus activating NK cells expanded in vitro and promoting the anti-tumor effect of NK cells,so as to provide theoretical basis and lay an experimental foundation for NK cells combined with reovirus in tumor therapy.Methods:1.Human peripheral blood mononuclear cells?PBMCs?was isolated from peripheral blood mononuclear cells of healthy blood donors,and inactivated K562 cells were used as feeder cells.The PBMCs were induced and cultured in vitro for about 14 days under the action of IL-2,and the purity was detected by flow cytometry.2.NK cells were treated with reovirus?Reo-NK?and ds RNA analogue poly?I:C??poly-NK?.The expression of CD69 on the surface of NK cells were detected by flow cytometry,the death rate of DLD-1cells were detected by CCK-8,and the secretion levels of perforin and interferon-??IFN-??were detected by ELISA.3.After Reo-NK and poly-NK were treated with TLR3/ds RNA complex blocker and TBK1/IKK?inhibitor BX795,the expression of NK cells activation marker CD69 were detected by flow cytometry,the death rate of DLD-1 cells were detected by CCK-8,and the secretion levels of perforin,IFN-?and TNF-?of Reo-NK and Poly-NK were detected by ELISA.Results:1.The purity of NK cells expanded in vitro can reach about 90%,and the survival rate is more than95%.2.TLR3/ds RNA complex inhibitor inhibits the activation of NK cells by reovirus,and reduces the positive rate of CD69+NK cells,inhibits the killing of DLD-1cells by NK cells,and reduces the secretion of IFN-?and perforin.?1?.The death rate of DLD-1 cells decreased:Treated with 10?m TLR3/ds RNA complex inhibitor of Reo-NK(Reo-NKinhibit.),the death rate of DLD-1 cells decreased compared with that of untreated Reo-NK[?49.58±2.1?%vs?65.6±1.3?%,**P<0.01];and in poly-NK(Poly-NKinhibit.),the cell death rate of DLD-1 cells was lower than that of untreated Poly-NK[?42.4±2.6?%vs?69.3±2.5?%,***P<0.001];?2?.TLR3/ds RNA complex inhibitor reduces the death rate of NK cells to DLD-1 cells;?3?.The secretion levels of TNF-?and IFN-?decreased after treated with 10?m TLR3/ds RNA complex inhibitor:a.TNF-?:Reo-NKinhibit.decreases with Reo-NK,[?268.3±2.1?pg/m L vs?201.3±3.2?pg/m L,*P<0.05];Poly-NKinhibit.decreases with Poly-NK[?231.2±2.12?pg/m L vs?189.3±2.2?pg/m L],*P<0.05.Similarly,the secretion of IFN-?under the action of TLR3/ds RNA complex inhibitor was also lower than that without TLR3/ds RNA complex inhibitor treatment.3.TBK1/IKK?inhibitor BX795 reduces the activation of ds RNA on NK cells,reduces the positive rate of CD69+NK cells,inhibits the killing of DLD-1 cells by NK cells,and reduces the secretion of IFN-?and perforin.?1?The expression of CD69?an activation marker on the surface of NK cells?:Reo-NK is higher than NK?63.1%vs 58.3%?;Reo-NK+Bx is lower than Reo-NK?46.1%vs 63.1%?;Poly-NK is higher than NK?70.5%vs 58.3%?,while Poly-NK+Bx decreased compared with Poly-NK:?60.0%vs 70.5%?;?2?The killing rate of NK on DLD-1 cells:compared with NK,the killing rate of Reo-NK was significantly higher than that of NK[?69.58±1.26?%vs?46.0±2.2?%,(**P<0.0l)],and the killing rate of DLD-1 cells treated with 10?m BX795 blocker?Reo-NK+Bx?was inhibited[?69.6±1.3?%vs?60.2±2.1?%,?*P<0.05?];The cytotoxicity of Poly-NK to DLD-1 cells was significantly higher than that of NK[?69.3±2.5?%vs?46.0±2.2?%,*P<0.001].The cytotoxicity of Poly-NK to DLD-1 cells decreased to?60.0±1.6?%after treatment with 10?M BX795 blocker?Poly-NK+Bx?.?3?The secretion levels of Perforin,IFN-?and TNF-?in the culture supernatant of NK cells were as follows:a.In the detection of perforin,the secretion level of Reo-NK was significantly higher than that of NK alone[?1120.0±10.9?pg/m L vs?825.0±10.9?pg/m L,*P<0.05],and the secretion level of Reo-NK+Bx treated with 10?M BX795was significantly lower than that of Reo-NK,[?838.0±8.3?pg/m L vs?1120.0±11.0?pg/m L,*P<0.05].The secretion level of Poly-NK was significantly higher than that of NK alone[?1008.5±5.3?pg/m L vs?825.0±10.9?pg/m L,*P<0.05].The secretion level of 10?M BX795 treatment?Poly-NK+Bx?was significantly lower than that of Poly-NK[(740.0±10.6 pg/m L vs?1008.5±5.3?pg/m L,**P<0.01];b.In the detection of IFN-?,the secretion level of Reo-NK was significantly higher than that of NK alone,and the secretion level of Poly-NK was also significantly higher than that of NK(**P<0.01).Compared with NK,the secretion level of Poly-NK was also significantly higher than that of NK.The secretion level of Poly-NK treated with10?M BX795?Poly-NK+Bx?was significantly lower than that of Poly-NK[?52.2±4.6?pg/m L vs?147.0±2.7?pg/m L,**P<0.01].Conclusion:1.Reovirus can directly promote the activation of NK cells,and the activation of NK cells is similar to that of Poly?I:C?;2.The molecular mechanism of reovirus activating NK cells may be related to TLR3 signal pathway. |
PDF Full Text Request