NDV-HN经非依赖IFN途径上调NK细胞TRAIL表达增强抗瘤作用的新机制研究

It is showed that Newcastle disease virus(NDV) can directly and indirectly kill tumor cells in vitro and in vivo, but its mechanisms are not completely clear. Natural killer (NK) cell is one of important killing cells, which can induce apoptosis of target cells through TNF-related apoptosis-inducing ligand-death receptor(TRAIL-DR) pathway. Our privious study shows that NDV can upregulate the TRAIL expression of NK cells through induction of IFN-γ,but TRAIL expression also can be detected on NDV-stimulated NK cell from IFN R-/- mice (fuctional inactivation of the IFN receptor gene, cells from IFNR-/- mice were insensitive to the antiviral activity of IFN).This result reminds us that it is likely that NDV can upregulate the TRAIL expression of NK cells through other pathways without IFN dependance. So we will use molecular biology and other techniques to perform the following work: ①Verify the primary binding site of NKp46 to virual HN through IFN-independance pathway. ②Demonstrate that Syk tyrosine kinase and NF-κB factor have taken part in the regulation of TRAIL in HN-stimulated NK cells through IFN-independance pathway. ③Investigate the effect on effectors and tumoricidal activity of NK cells on hepatoma cell lines by inducing or blocking of molecular involved in IFN-independence pathway. This project will further elucidate the mechanism that NDV kills tumor cells and will provide the new foundamentional theory for the aplication of anti-tumoral therapy based on NDV-HN- stimulated NK cell transfusion.

我们前期的研究工作证实了NDV-HN可通过诱导IFN-r 分泌上调NK细胞TRAIL表达来增强抗瘤作用，然而IFN受体缺陷小鼠的NK细胞经NDV-HN刺激后TRAIL仍能上调表达，此结果提示可能存在NDV-HN经"非依赖IFN途径"上调NK细胞TRAIL表达的新机制，国内外对此尚无报道。所以本项目拟以"非依赖IFN途径"为研究切入点，用酵母双杂交等技术方法开展如下研究：首先明确NK细胞在"非依赖IFN途径"下识别并结合的NDV-HN的氨基酸位点，接着研究参与NK细胞"非依赖IFN途径"上调TRAIL的胞内信号调控分子，最后探讨"NDV-HN-TRAIL"信号通路中关键分子经刺激或阻断处理后效应NK细胞膜上TRAIL等功能性表型及对肝癌细胞杀伤的变化。项目的完成可望进一步阐明NDV-HN活化NK细胞的抗肿瘤免疫机制，也为基于"NDV-HN-NK细胞"输注的肿瘤生物治疗提供新的理论依据。

新城疫病毒（Newcastle disease virus，NDV）抗肿瘤的免疫活化机制是目前肿瘤治疗研究的新热点之一。NDV的抗瘤效果不仅与直接诱导肿瘤细胞的凋亡和自噬有关，还与NDV激活机体抗瘤免疫应答有关。我们前期工作证实NDV-HN可诱导IFN-γ分泌上调NK细胞TRAIL表达来增强抗瘤作用，然而IFN受体缺陷小鼠的NK细胞经NDV-HN刺激后TRAIL仍能上调，提示可能存在"非依赖IFN途径"的NDV活化新机制。本项目以"非依赖IFN途径"为切入点，获得如下发现：（1）NDV存在IFN-γ非依赖途径，且进一步证实HN-NKp46途径是NDV不依赖IFN-γ促NK细胞TRAIL上调的活化途径之一；（2）Syk、NF-κB信号通路在HN经HN-NKp46途径上调TRAIL的表达中起重要作用；（3）HN-NKp46活化通路还参与调节NK细胞其他重要抗瘤分子GrB和Fas/FasL的释放和表达；（4）NDV-HN具有活化小鼠IFN R-/-NK细胞所致的小鼠肝癌细胞杀伤的能力。这对进一步阐明NDV-HN直接活化NK细胞的分子机制，对应用NDV溶瘤病毒合并NK细胞于肿瘤的生物治疗具有积极的意义。