基于HIF-1α/VEGF信号通路研究天冬胶抗肝癌血管生成的机制

Angiogenes induced by hypoxia is one of predomiant phenotypes of primary liver cancer ,features that could be important in the development of new interventional embolization drugs. HIF-1α/ VEGF signaling pathway is activated in hypoxia and related to features of malignancies. Recently, we found that asparagus colloid made from asparagus polysaccharides is a good performance of embolic agents. It can bring anti-cancer efficacy after treatment of liver cancer by hepatic artery embolization, and show little toxicity to the host. Interestingly, our preliminary data indicate that refined asparagus colloide can significantly low HIF-1α、VEGF’s expression to a certain extent. We hypothesize that HIF-1α may be the key of molecular-targeted of asparagus polysaccharides.This project is designed to investigate the functions of HIF-1α/ VEGF signaling pathway in live tumor progression, combined with gene transfection, RNA interference, RT-PCR, immunoprecipitation.We will determine the contribution of Asparagus polysaccharides to tumor cell proliferation,survival,migration and invasion due to hypoxia. We will also investigate the underlying molecular mechanisms,mainly focused on HIF-1α/ VEGF signaling pathway.Then we will study the biological roles of HIF-1α/ VEGF signaling pathway in live tumor proliferation, invasion and metastasis using a xenograft animal model. Thus, this proposed study will characterize the novel roles and pathways of HIF-1α/ VEGF signaling pathway in live cancer progression. The information gained should be of help in developing new therapeutic approaches to treat malignant liver tumour.

缺氧诱导血管生成是肝癌的重要生物学特征。HIF-1α/VEGF信号通路在缺氧下激活并与肝癌的预后关系密切。我们前期发现，天冬多糖制成胶剂在大鼠肝动脉栓塞治疗肝癌具有较好的疗效，且毒性低。进一步研究发现缺氧下天冬多糖处理的肝癌细胞HIF-1α、VEGF表达明显下调，但具体机制不详。我们推测，天冬胶介入治疗肝癌可能是直接调控HIF-1α或影响其上、下游基因，来调控HIF-1α/VEGF信号通路，从而抑制肿瘤血管生成及侵袭转移。本项目拟制备HIF-1α基因过表达和敲除的人肝癌细胞模型以及裸小鼠移植瘤模型，运用过表达、RNA干扰、免疫沉淀等技术手段，基于HIF-1α/VEGF信号通路系统研究天冬多糖抑制肿瘤血管生成、增殖、侵袭和转移。从基因、蛋白质和功能诸方面揭示天冬胶介入栓塞治疗肝癌的机制，阐明其抗血管生成的信号通路，为提高中药TACE治疗肝癌的临床疗效提供实验依据。

原发性肝癌（简称肝癌）是一种发病率高，发生、发展复杂的恶性肿瘤。作为富血管的实体瘤，肝癌的血管生成是其TACE临床治疗的最大障碍。缺氧是肿瘤重要的微环境，缺氧导致缺氧诱导因子-1α（HIF-1α）的表达升高，并诱导下游基因产物如胰岛素样生长因子II和血管内皮细胞生长因子（VEGF）的高表达，它们是支持肿瘤生长的重要因素。本项目前期研究发现，天冬胶介入治疗肝癌可调控血管生成，在此基础上，我们首先利用公共信息数据库探索了HIF-1α在临床样本中的表达和预后差异，并在获得纯度为98%的天冬多糖后，我们用HIF-1α过表达和HIF-1αRNAi慢病毒感染肝癌细胞，在常氧和缺氧（模拟精制天冬胶介入治疗状态）条件下，发现天冬多糖可以抑制肝癌细胞增殖、迁移、侵袭及血管形成，机制研究发现天冬多糖在常氧下和缺氧下均可抑制肝癌细胞p-mTOR、p-AKT、p-ERK蛋白表达，而对mTOR、AKT和ERK蛋白表达影响不明显。加入PI3K激动剂和MAPK激动剂后，研究发现PI3K激动剂和MAPK激动剂可以逆转天冬多糖对肝癌细胞迁移、侵袭和血管形成作用，并可逆转天冬多糖对p-mTOR、p-AKT、p-ERK、p38的表达，验证了天冬多糖抑制肝癌细胞增殖、迁移、侵袭及血管形成的功能是通过PI3K和MAPK信号通路实现的，体内应用裸鼠移植瘤模型实验结论与体外实验结论一致。最后我们继续研究了HIF-1α RNAi联合天冬多糖对肝癌细胞的抑制作用，进行体内和体外实验继续探索天冬多糖联合HIF-1α 过表达的协同作用对肝癌细胞的增殖、迁移、侵袭和血管形成的影响，为将天冬多糖制成胶样制剂（精制天冬胶），通过介入放射学的方法（瘤内注射、动脉灌注栓塞）治疗恶性肿瘤提供理论依据。