基于钠离子通道β亚基的蝎镇痛抗肿瘤缬精甘肽抗肝癌作用机制初探

Hepatocellular Carcinoma (HCC) is the third worst disease in the world with a highly incidence rate in China. The peptide from Buthus martensii Karsch (BmK) is a valuable drug which has the potential antitumor activity. Now it is still dimness that the mechanism between the antitumor-analgesic peptide (AGAP) and the voltage-gated Na+ channel β subunits. Our laboratory has expressed AGAP in E.coli successfully, definited that hepatoma carcinoma cell line with various immigration, such as Hep 3B, Bel-7402, Hep G2, expressed specified β subunits. So we proved that β subunits are connected to the immigration of cells. According to the various expression of β subunits of three strain cells, a series of in vivo experiments including transfection and RNAi were performed. We examined cells multiplication, immigration and invasion after applied AGAP. The purpose of this project is to look for the drug target site and build the screening model of anti-hepatocellular carcinoma drug through researching the relationship between AGAP and the voltage-gated Na+ channel β subunits, explore the mechanism of this peptide in the level of ion channel and develop the variety of Traditional Chinese Medicine with independent intellectual property rights. The research have provided evidence and laid a good foundation for further experiment.

肝癌是世界第三大癌症，在中国发病率极高。东亚钳蝎是一种宝贵的来源于药用动物的潜在抗肿瘤药物。目前，关于蝎镇痛抗肿瘤缬精甘肽与肿瘤电压门控钠离子通道β亚基间的作用机制尚不清楚。前期工作已在大肠杆菌表达具有抗肿瘤活性的重组蝎镇痛抗肿瘤缬精甘肽；确定迁移能力不同的肝癌细胞株(Hep 3B，Bel-7402，Hep G2)差异表达特定电压门控钠离子通道β亚基；证明电压门控钠离子通道β亚基与肝癌细胞的迁移相关。本项目针对电压门控钠离子通道β亚基表达存在差异性，分别对三株肝癌细胞进行转染或RNA干扰实验，蝎镇痛抗肿瘤缬精甘肽作用后检测细胞增殖、迁移及侵袭能力变化。研究蝎镇痛抗肿瘤缬精甘肽同肝癌细胞电压门控钠离子通道β亚基作用的相关性，进而寻找药物作用靶点并建立抗肝癌药物筛选模型，从离子通道水平探索蝎镇痛抗肿瘤缬精甘肽的作用机制，开发拥有原创性自主知识产权的中药新品种等方面，奠定必要的物质基础。

在我国，肝癌发病位于恶性肿瘤的第二位，肝癌死亡率位居我国消化道恶性肿瘤死亡率之首。近年来研究发现，电压门控钠离子通道在肿瘤的发生发展过程中发挥着重要作用，本课题就电压门控钠离子通道在HepG2细胞中的表达以及对细胞迁移、侵袭和增殖的影响展开研究。.本课题主要开展下列三个方面的研究工作：(Ⅰ)利用RT-PCR及Western Blot技术，检测电压门控钠离子通道β亚基在不同迁移能力的肝癌细胞中的表达，结果表明迁移性较强的肝癌细胞Hep3B中有β1亚基的微量的表达，迁移性较弱的HepG2细胞中则有β1和β3亚基的表达，而正常肝细胞中则没有β亚基的表达，因此不同肝癌及肝细胞中表达有不同的电压门控钠离子通道β亚型；(II)以表达有两种β亚基的HepG2细胞为研究对象，利用RNAi及流式细胞术等技术，检测β亚基对细胞迁移、侵袭及增殖的影响，结果表明：β1亚基主要影响HepG2细胞的迁移及侵袭，而β3亚基主要影响细胞的增殖并诱导细胞凋亡；(III) rAGAP为本实验室重组表达的镇痛抗肿瘤缬精甘肽，其剂量依赖性的抑制HepG2细胞的迁移、侵袭及增殖，并伴随着β1亚基表达的上调及β3亚基表达的下调，提示rAGAP可能通过影响HepG2细胞中β亚基的表达水平抑制细胞的迁移、侵袭、增殖并诱导细胞凋亡。