蟾毒配基纳米制剂抗脑胶质瘤作用及机制的研究

The incidence of glioma was the highest in brain tumors, but the chemotherapeutic drugs, with the ability to kill the glioma cells, didn't show satisfactory treatment effectiveness because of their low blood brain barrier permeability. Toad venoms and their preparations had long history on the treatment of tumors, but there was rare study on their activities against brain glioma. In the previous study, we found that bufadienolides could cross through blood brain barrier easily and exhibit high cytotoxic effect on U87-MG cells, both effects of which were further increased when bufadienolides nano preparation (BU-NP) was prepared.In addition, we also found that BU-NP could increase the generation of reactive oxygen species (ROS) in the U87-MG cells. Based on these results, in the present study, orthotopic implantation model of brain glioma cells in nude mice, magnetic resonance image, transmission electron microscopy and immunohistochemistry technology were used to study the anti-brain glioma activity of BU-NP in vivo; immunofluorescence, confocal laser scanning microscopy, flow cytometry, western blot and immune electron microscopy were used to study the cell death type and specific molecular mechanism through ROS-JNK signalling pathway mediated by BU-NP. The study will bring a new breakthrough on the treatment effectiveness and molecular mechanism of traditional Chinese medicine ingredients of bufadienolides against brain gliomas, and at the same time, lay the theoretical foundation of BU-NP as a new drug for the treatment of brain gliomas.

脑肿瘤中胶质瘤发病率最高，但药物治疗效果差强人意，很大原因是由于能杀伤胶质瘤细胞的药物很难同时具有穿过血脑屏障的能力。蟾酥及其制剂治疗肿瘤历史悠久，但鲜有研究报道其对脑胶质瘤的作用。前期研究发现蟾毒配基易透过血脑屏障，对脑胶质瘤U87-MG细胞有很强的细胞毒性，制备成纳米制剂（BU-NP）后，脑靶向性以及对U87-MG细胞的生长抑制作用更强。此外，还发现蟾毒配基可增高细胞内ROS含量。基于此，本课题采用脑胶质瘤裸鼠原位移植瘤模型、核磁共振成像、透射电镜、免疫组化技术，研究BU-NP体内抑制脑胶质瘤生长的作用；采用免疫荧光、激光共聚焦扫描、流式细胞术、免疫印迹、免疫电镜方法研究BU-NP增高ROS激活JNK诱导的U87-MG细胞死亡方式及具体的分子机制。完成本课题对蟾毒配基这一传统的天然药用成分抗胶质瘤的作用和机制会有一个新的突破，同时为将BU-NP开发成治疗胶质瘤的新型药物奠定理论基础。

脑肿瘤中胶质瘤发病率最高，但药物治疗效果差强人意，很大原因是由于能杀伤胶质瘤细胞的药物很难同时具有穿过血脑屏障的能力。蟾酥及其制剂治疗肿瘤历史悠久，但鲜有研究报道其对脑胶质瘤的作用。前期研究发现蟾毒配基易透过血脑屏障，对脑胶质瘤U87-MG细胞有很强的细胞毒性，制备成纳米制剂（BU-NP）后，脑靶向性以及对U87-MG细胞的生长抑制作用更强。此外，还发现蟾毒配基可增高细胞内ROS含量。基于此，本课题继续深入研究，采用脑胶质瘤细胞裸鼠原位移植动物模型以及U87-MG细胞分别进行BU-NP的体内外抑瘤实验及机制研究，结果表明：BU-NP在体内外均呈剂量依赖性抑制脑胶质瘤生长，并且等剂量的BU-NP比游离蟾毒配基（BU-S）作用更强；脑胶质瘤组织及细胞中ROS的含量、p-JNK蛋白以及与凋亡自噬相关的蛋白（cleaved-caspase-3、cleaved-caspase-9、LC3-II和Beclin-1）的表达均随BU-NP给药剂量的增加而增加，表明BU-NP是通过诱导细胞凋亡和过度自噬两种方式共同杀伤脑胶质瘤细胞的，且在杀死细胞过程中JNK被激活并伴随有ROS产生；抗氧化剂预先处理U87-MG细胞后，可同时降低BU-NP诱导的p-JNK表达及ROS产生，而JNK抑制剂只能降低BU-NP诱导的p-JNK表达，而对ROS含量变化没有影响，表明在BU-NP诱导的细胞死亡过程中ROS是JNK的上游，BU-NP是通过先增加ROS然后激活JNK诱导细胞死亡的。本课题证明了蟾毒配基纳米制剂体内外抑制脑胶质瘤生长的作用并且阐明了其作用机制，使我们对蟾毒配基这一传统的天然药用成分抗脑胶质瘤的作用和机制有了一个全新的认识，为将BU-NP开发成治疗胶质瘤的新型药物奠定理论基础。