天然溶瘤病毒M1靶向性杀伤恶性脑胶质瘤细胞的胞内机制研究

Human malignant glioma is a disease with high degree of malignancy, extensive invasion, short course, high death rate, at the same time without effective therapy. The oncolytic virus may specifically kill tumor cells without any harm to normal cells. It has been proved that alphavirus M1 target a variety of glioma cells， induce apoptosis and has a good oncolytic effect on the rat orthotopic glioma model rats. The oncolytic mechanism of alphaviruses, which is inhibiting the intracellular translation, has been gradually been accepted as a consensus. However, for Alphavirus, the reason why it exclusively target tumor cells is largely unknown. According to our results, we proposed an intracellular mechanism underling the tumor-specificity: with PTEN deficiency in most glioma cells, down-regulation activity of miR-29a on PTEN can not work, leaving only its down-regulation activity on PI3K, thereby promoting cell to die. Firstly，this study uses drugs such as LY294002, rapamycin to get the conclusive evidence of the PI3K/AKT pathway in M1 infection, and its impact on viral replication and apoptosis, and uses the WB, the reporter gene analysis method to determine the targeted down-regulation of miR-29a on PI3K and PTEN. The project will provide a scientific basis for clarifing the tumor targeted mechanism of alphavirus; promote M1 developing as an oncolytic virus, and provide theoretical support to the individualized therapy.

脑胶质瘤恶性程度强、易侵袭、病死率高，迄今无良药可医。溶瘤病毒因可靶向性杀伤肿瘤细胞而倍受重视。我们发现甲病毒M1可靶向性杀伤胶质瘤细胞，而无害于正常细胞，明显延长胶质瘤大鼠的存活。翻译抑制虽为甲病毒共认的杀瘤机制，但其专杀肿瘤细胞的嗜瘤机制不明。我们发现M1感染后（可靶向PI3K和PTEN的）miR29a上调，众所周知PTEN在胶质瘤细胞中多有变异，而正常细胞中无变异，故极可能"在PTEN变异的细胞中，miR29a不能平衡地下调PI3K和PTEN，细胞因'PI3K促活通路'被阻断而死亡"。本研究拟运用基因沉默、过表达等手段确认PI3K通路被阻断在M1嗜瘤机制中的意义，并用WB、报告基因等方法确定miR29a如何双向调控PI3K通路，最后通过干预miR29a确证其在M1嗜瘤机制中的关键作用。本项目对甲病毒专杀肿瘤细胞的嗜瘤机制之阐明将为把其改造成更高效、更特异的溶瘤病毒奠定坚实的理论基础。

背景：.常规放化疗在应用上具有局限性，肿瘤医学临床上急需发现能够靶向肿瘤的新疗法。溶瘤病毒因可靶向性杀伤肿瘤细胞而倍受重视。我们发现甲病毒M1 可靶向性杀伤胶质瘤、肝癌、结直肠癌等多种肿瘤细胞，而无害于正常细胞，明显延长荷瘤鼠的存活，但其专杀肿瘤细胞的嗜瘤机制不明。本研究拟确定甲病毒M1 专杀肿瘤细胞的嗜瘤机制。.研究内容与重要结果（关键数据）：.研究鉴定了甲病毒M1对不同类型肿瘤组织的靶向性溶瘤效应。我们体外细胞实验及动物体内实验证明甲病毒M1可以特异性靶向胶质瘤、肝癌、结直肠癌、黑色素瘤等多种肿瘤细胞进行溶瘤，而不破坏正常细胞。.研究揭示了甲病毒M1靶向肿瘤组织的特异性分子机制。我们发现肿瘤细胞普遍存在的抗病毒ZAP蛋白低表达是其易受甲病毒M1攻击的主要原因之一。.研究揭示了甲病毒M1破坏杀伤肿瘤组织的效应性分子机制。我们实验发现，甲病毒M1感染可以导致被感染肿瘤细胞发生相较于被感染正常细胞更为严重的内质网应激，并由此引起不可逆转的细胞凋亡。.研究发现了甲病毒M1溶瘤效应的增效剂。我们研究发现cAMP通路或EPAC通路激活剂对甲病毒M1溶瘤效应的增效作用。此增效作用并不影响溶瘤效应特异性。.研究证实了甲病毒M1应用的非人灵长类动物体内安全性。我们通过静脉注射溶瘤病毒M1入食蟹猴体内，通过生物化学、免疫学、影像学及临床观察等全面确证了其体内安全性。.科学意义：.本项目对此嗜瘤机制、效应机制之阐明，增效剂的发现及非人灵长类动物实验结果将为把其改造成更高效、更安全的溶瘤病毒奠定坚实的理论基础。