论文部分内容阅读
目的:研究新城疫病毒(Newcastle disease virus,NDV)刺激自然杀伤(natural killer,NK)细胞产生肿瘤坏死因子相关凋亡诱导配体(TNF-related apoptosis-inducing ligand,TRAIL)的作用机制。方法:采用免疫磁珠分选(magnetic activated cell sorting,MACS)法,分离得到高纯度的NK细胞;乳酸脱氢酶释放法检测NDV激活的NK细胞对人肝癌HepG2细胞的杀伤作用;实时荧光定量-PCR(real-time fluorogenic quantitative-PCR,RFQ-PCR)检测NDV刺激NK细胞4h后对TRAILmRNA表达水平的影响;FCM检测NDV刺激NK细胞16h时TRAIL蛋白的表达情况;ELISA法检测NDV刺激NK细胞后对其分泌干扰素γ(interferon-γ,IFN-γ)的影响;最后采用抗IFN-γ抗体中和IFN-γ后,再采用FCM检测NDV刺激的NK细胞中TRAIL蛋白的表达情况。结果:FCM检测结果显示,分离得到的NK细胞的纯度达(90.60±1.15)%;NDV刺激NK细胞16h后,明显增强NK细胞的杀伤肿瘤作用,NDV效价为204.8HU刺激组的杀伤率达到(22.28±0.84)%;RFQ-PCR及FCM检测结果显示,不同效价的NDV刺激NK细胞后,NK细胞中TRAILmRNA及蛋白表达水平均明显升高;ELISA检测结果显示,NDV刺激NK细胞后IFN-γ的分泌水平明显增加,在NDV效价为25.6HU刺激16h时达到峰值(796.47±37.87)pg/mL;采用抗IFN-γ抗体中和IFN-γ后,NK细胞中TRAIL蛋白表达水平明显下降。结论:NDV刺激NK细胞后可增强IFN-γ的分泌水平,进而通过IFN-γ上调TRAIL的表达,此途径为NDV刺激NK细胞表达TRAIL的机制之一。此外,NDV直接刺激NK细胞表达TRAIL是另一条重要的途径。
Objective: To investigate the mechanism of Newcastle disease virus (NDV) on the stimulation of natural killer (NK) cells to produce tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Methods: High purity NK cells were isolated by magnetic activated cell sorting (MACS) method. The killing effect of NDV-activated NK cells on HepG2 cells was detected by lactate dehydrogenase release assay. Real- The expression of TRAIL mRNA in NDV-stimulated NK cells was detected by real-time fluorogenic quantitative-PCR (RFQ-PCR). The expression of TRAIL protein in NDV-stimulated NK cells at 16h was detected by FCM. IFN-γand IFN-γsecreted. Finally, the expression of TRAIL protein in NDV-stimulated NK cells was detected by FCM after neutralizing IFN-γwith anti-IFN-γ antibody. Results: FCM results showed that the purity of isolated NK cells was (90.60 ± 1.15)%. After NK cells were stimulated by NDV for 16 hours, the cytotoxicity of NK cells was significantly enhanced. The killing rate of NDV with 204.8HU stimulation group reached (22.28 ± 0.84)%, respectively. The results of RFQ-PCR and FCM showed that the expression of TRAIL mRNA and protein in NK cells were significantly increased after NK cells were stimulated with different titer of NDV. ELISA assay showed that NDV stimulated NK cells IFN The level of TRAIL protein expression in NK cells was significantly increased after IFN-γ was neutralized by anti-IFN-γ antibody (P <0.05), which was significantly increased after NDV titers of 25.6HU stimulated for 16h (796.47 ± 37.87) pg / decline. Conclusion: NDV stimulated the NK cells to enhance the secretion of IFN-γ, and then upregulated the expression of TRAIL through IFN-γ, which is one of the mechanisms by which NDV stimulated NK cells to express TRAIL. In addition, NDV directly stimulates NK cells to express TRAIL is another important pathway.