仙台病毒诱生IFN-β干扰SCID小鼠黑色素瘤模型建立的分子机制

Hemagglutinating virus of Japan (HVJ) can induce interforn-β (IFN-β), causing apoptosis in tumor cells and interfering with the establishment of transplanted tumor models in mice. In addition to the Janus kinases/signal transducers and activators of transcription (JAK/STAT) signaling pathway, there are other evidences demonstrating that IFN-β induced by HVJ could induce apoptosis in cancer cells by still unknown signaling pathways. Our previous study indicated that HVJ-induced IFN-β could induce apoptosis in murine melanoma cells, and the down-expression of insulin-like growth factor-1 receptor (IGF-1R) by HVJ in the cells was first discovered. However, the specific mechanism to this remains unknown. To further investigate the mechanism of apoptotic induction by HVJ in tumor cells，in this project, interferon α/β receptor antibody or siRNA specific to interferon-β promoter stimulator 1 (IPS-1) is going to be used to block the type I interferon signaling pathway, and JAK inhibitor will be used to block the JAK/STAT signaling pathway. Through the detection of IFN-β and IGF-1R expression, the relationship between HVJ-induced IFN-β and the IGF-1R expression is aiming to be elucidated. In addition, fluorescence-activated cell sorter (FACS), immunohistochemistry and immunoblot analysis will be carried out to investigate the effect of HVJ-induced IFN-β on phosphatidylinositol 3-kinase/Serine/threonine Kinase (PI3K/Akt) and mitogen-activated protein kinase (MAPK) signaling pathways in vitro and in vivo. Based on the above studies, this research has the potential to throw light on new mechanisms in apoptosis induction by HVJ-induced IFN-β in tumor cells, and provide scientific evidences to further elucidate the mechanisms in interfering with the establishment of tumor models by HVJ.

仙台病毒(HVJ)诱生IFNβ可致肿瘤细胞凋亡，并干扰小鼠移植瘤模型建立。研究证实，除JAK/STAT外，HVJ诱生IFNβ一定存在其他未知信号通路可致肿瘤细胞凋亡。本项目前期研究表明：HVJ诱生IFNβ可致小鼠黑色素瘤细胞凋亡，并首次证实其中的胰岛素样生长因子1受体(IGF-1R)表达下降，但具体的调控机制尚未阐明。为进一步研究HVJ致肿瘤细胞凋亡的分子机制，本项目以IFNβ为切入点，用IFNα/β受体抗体或siRNA阻断I型干扰素信号通路，并用JAK抑制剂封闭JAK/STAT通路，阐明HVJ诱生IFNβ对IGF-1R表达的调控；并通过流式细胞术、免疫印迹和免疫组化等手段，从体内、外两个层面，研究HVJ诱生IFNβ对IGF-1R下游调控的PI3K-Akt和MAPK信号通路的影响，旨在明确HVJ诱生IFNβ致肿瘤细胞凋亡的新途径，为进一步阐明HVJ干扰小鼠移植瘤模型建立的机制提供科学依据。

仙台病毒感染啮齿类实验动物可从细胞凋亡等方面导致多种移植瘤模型制备失败，对科学研究和药物研发产生重大干扰。本研究以小鼠黑色素瘤细胞（B16F10）为模型，从细胞形态、增殖活性和凋亡水平等途径研究了仙台病毒干扰小鼠黑色素瘤模型建立的作用机制。结果显示，仙台病毒可剂量依赖性地诱导B16F10凋亡并抑制小鼠黑色素瘤细胞生长，直接在荷黑色素瘤裸鼠瘤内注射仙台病毒，可见仙台病毒可抑制IGF-1R蛋白表达。为深入阐明仙台病毒诱导B16F10凋亡的机制，免疫印迹实验结果显示，仙台病毒可激活caspase-9，进而裂解caspase-3和PARP，从而引起细胞凋亡，且caspase激活而引发的细胞凋亡由MAPK途径调控，研究证实，经仙台病毒处理的B16F10中的ERK1/2，JNK，和p38 MAPK均被磷酸化，且p38 MAPK抑制剂SB203580、Jnk抑制剂SP600125和MEK1/2抑制剂U0126也验证了仙台病毒能诱导B16F10细胞的MAPK通路激活，说明MAPK通路在仙台病毒诱导B16F10凋亡中起重要作用。更有趣的是，本研究发现仙台病毒诱生I型干扰素在致B16F10凋亡中只发挥部分作用，而β干扰素启动子刺激分子1(IPS-1)在仙台病毒干扰小鼠黑色素瘤模型建立过程中发挥着双重作用。最后通过裸鼠皮内接种B16F10细胞制作小鼠黑色素瘤模型，小鼠瘤内注射仙台病毒样粒子，治疗结果表明仙台病毒治疗组小鼠的肿瘤体积显著小于PBS对照组，且在成瘤后40天内，仙台病毒治疗组小鼠的存活率为80%，而对照组为0%。免疫组化和TUNEL检测结果显示，仙台病毒治疗组肿瘤细胞崩解萎缩，并形成大量细胞凋亡，表现为核浓缩，固裂。据此，本研究以体内、外模型，从免疫调节、凋亡等信号通路等方面揭示了仙台病毒干扰小鼠黑色素瘤模型建立的分子机制。