| Part1:Effects of NDV stimulation in vitro on TRAIL expression of mouse spleen NK cellsObjective:To investigate the expression levels of TRAIL in mouse spleen natural killer (NK) cells after NDV stimulation in vitro.Methods:Spleen NK cells were obtained from BALB/c mice by MACS separator.NK cells were stimulated for12h with25HU/ml NDV7793in DMEM medium.TRAIL mRNA expression in NK cells was analyzed through RT-PCR assay. The protein expression of TRAIL in NK cells was detected by Western blotting analysis. Furthermore, the expression of cell surface TRAILwas determined by flow cytometry, and that of soluble TRAIL was determined by ELISA assay in NK cells.Results:TRAIL was up-regulated at both the mRNA and protein levels in NK cells after NDV stimulation in vitro.Conclusion:NDV stimulation in vitro could enhance TRAIL expression in mouse spleen NK cells.Part2:NDV stimulation enhances killing activity of mouse spleen NK cells toward mouse hepatoma cell lines in vitro and its mechanisms.Objective:Toevaluate whether the tumoricidal activity of NK cells is enhanced with TRAIL up-regulation induced by NDV stimulation, and to investigate what is the mechanism of TRAIL regulation in the NDV-stimulated NK cells.Methods:Spleen NK cells were obtained from BALB/c mice by MACS separator.NK cells were stimulated for12h with25HU/ml NDV7793in DMEM medium. LDH method was used to quantify the cytotoxic activities of NK cells against mouse hepatoma cells (Hepal-6and Novikoff). IFN-y or soluble TRAIL (sTRAIL) concentrations in the supernatants of NK cells medium were determined by specific ELISA assay.The expression of cell surface TRAIL was determined by flow cytometry.Results:NDV stimulation enhanced the IFN-γ secretion and tumoricidal activity of NK cells toward mouse hepatoma cell lines (Hepal-6and Novikoff) in vitro. Treating with anti-TRAIL neutralizing Ab induced significant decline in the cytotoxicity of NK cells toward Novikoff cell line (P＜0.01). After treating with anti-IFN-γ neutralizing Ab at the concentration of6μg/ml, significant suppressions in the production of soluble TRAILand surface expression of TRAIL were observed in NDV-stimulated NK cells (P＜0.05). Moreover, anti-IFN-γ neutralizing Ab down-regulated the tumoricidal activity of NDV-stimulated NK cells (P＜0.01).Conclusion:NDV stimulation could trigger the tumoricidal activity of NK cells by TRAIL induction through the IFN-y pathway.Part3:Intraperitoneal injection with NDV affects miceperipheral blood levels of TRAIL and tumoricidal activity of spleen NK cells Objective:To investigate the effects of NDV intraperitoneal injection on TRAIL expression in mice peripheral blood and spleen NK cell cytotoxicity toward hepatoma cells.Methods:NDV wasinjected (25HU/g) to BALB/c mice through intratumoral route. Twelve hours after injection, killed mice and collected peripheral blood. The concentrations of IFN-y and TRAIL in peripheral blood were determined by specific ELISA kits. Mice spleen NK cells were separated by MACS isolation kit. The mRNA level and protein expression of TRAIL in NK cells was analyzedthrough RT-PCR and Western blotting analysis.LDH method was used to quantify the cytotoxic activities of NK cells against Novikoff hepatoma cell line.Results:NDV intraperitoneal injectioninduced significant up-regulation of IFN-y and TRAIL concentrations in the peripheral blood of BALB/c mice, compared to the control group (P＜0.01and P＜0.05, respectively). Intraperitoneal injection with NDV enhanced TRAIL expression at both mRNA and protein levels in mouse spleen NK cells. Furthermore, intraperitoneal injection with NDV enhanced tumoricidal activity of spleen NK cells toward No vikoff hepatoma cell line in vitro. In blocking test, either anti-TRAIL neutralizing Ab or anti-TNF-αneutralizing Ab combined with anti-FasL neutralizing Ab could inhibit tumoricidal activity of NK cells (P＜0.01). But, the extent of inhibition on NK cells tumoricidal activity was greater with combination of anti-TNF-αneutralizing Ab and anti-FasL neutralizing Ab than that single use of anti-TRAIL neutralizing Ab (P＜0.01). The most significant suppression in the tumoricidal activity of NK cells was observed when combining all three neutralizing Abs (anti-TRAIL, anti-TNF-α and anti-FasL), and the suppression rate was78.2%(P＜0.01).Conclusion:Intraperitoneal injection with NDV could up-regulate mice peripheral blood levels of TRAIL and tumoricidal activity of spleen NK cells. But TRAIL was not the unique effectormoleculewhich mediates the tumoricidal activity in spleen NK cells that activated in vivo.Part4:Intraperitoneal administration of NDV reduces tumor growth in a H22hepatoma xenograft mouse model and its mechanismObjective:Toinvestigate the anti-tumor effect of NDV strain7793on H22hepatoma xenograft in BALB/c mice and explore the mechanismof this virotherapy.Methods:H22malignant ascites was implanted into BALB/c mice by subcutaneous injection to establish xenograft model. NDV was injected to mice through intraperitoneal route. The growth of xenograft and physique of mice was monitored daily. Twenty eight days later after ascites injection, mice were killed and peripheral blood was collected.The concentrations of IFN-y and TRAIL in peripheral blood were determined by specific ELISA kits. The xenograft tumor was removed and weighed to calculate tumor growth inhibition rate.Mice spleen NK cells were separated by MACS isolation kit. The mRNA level and protein expression of TRAIL in NK cells was analyzedthrough RT-PCR and Western blotting analysis. LDH method was used to quantify the cytotoxic activities of NK cells against Novikoff hepatoma cell line.Results:Six days after subcutaneous injection of H22malignant ascites the xenograft model in BALB/c mice was successfully established.NDV7793 intraperitonealinjection showed inhibitory effect on tumor growth, the tumor growth inhibition rate was52.6%. Tumor regression was observed in3mice (18.8%), and in one of these mice the tumor tissue regressed completely (6.3%). Survival analysisrevealed mouse survival rate in NDV group was significantly higher than that of control group (P＜0.01). Compared to the control group, the concerntrations of IFN-y and TRAILin peripheral blood of tumor bearing mice in NDV group were significantly higher (both P＜0.01). Intraperitoneal injection with NDV enhanced TRAIL expression at both mRNA and protein levels in mouse spleen NK cells.Furthermore, intraperitoneal injection with NDV enhanced tumoricidal activity of spleen NK cells toward Novikoff hepatoma cell line in vitro.In blocking test, neither anti-TRAIL neutralizing Ab nor anti-TNF-αneutralizing Ab combined with anti-FasL neutralizing Ab could inhibited tumoricidal activity of NK cells significantly (P＞0.05). The significant suppression in the tumoricidal activity of NK cells toward Novikoff hepatoma cell linewas observed only while combining all three neutralizing Abs (anti-TRAIL, anti-TNF-α and anti-FasL)(P＜0.01).Conclusion:Intraperitoneal injection with NDV has the capability to cause tumor regression of H22hepatoma xenograft in BALB/c mice through NK cells mediated antitumor immune response. TRAIL protein expressed by spleen NK cells was involved in this antitumor immune response. |
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