基于自噬天冬多糖下调肝癌HIF-1α作用机制研究

Hypoxia is the basis for the failure treatment of liver cancer，so that anti-angiogenesis is the key to the treatment of liver cancer in hypoxic microenvironmental. 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 decrease expression of HIF-1α and VEGF ,and promote apoptosis. Autophagy is activated in hypoxia which could promote HCC resistance to chemotherapy and antianiogenesis，and related to of malignancies.We hypothesize that autophagy may be the key of molecular-targeted of asparagus polysaccharides and interact with anti-angiogenesis, apoptosis to resist the response of liver cancer cells to hypoxia microenvironment. This project is designed to investigate the functions of autophagy in live tumor progression ,invasion and metastasis using liver cell in hypoxia and a xenograft animal model,combined with RNA interference.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 autophagy. Thus, this proposed study will characterize the novel roles and autophagy in live cancer progression. The information gained should be of help in developing new therapeutic approaches to treat malignant liver tumour.

缺氧缺血是肝癌治疗失败的基础。抗血管新生是治疗缺氧微环境肝癌的关键。前期我们研究中药提取物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可促进细胞凋亡，下调肝癌细胞HIF-1α及VEGF的表达，但缺氧下自噬的异常激活能抑制肝癌细胞凋亡、减低抗血管生成药物的敏感性，与肝癌的侵袭转移密切相关。由此提出假设，天冬多糖下调HIF-1α可能依赖调控自噬，及其与抗血管生成相互作用，从而抵抗肝癌细胞对缺血缺氧微环境的应答。本项目拟制备低氧下人肝癌细胞模型以及裸小鼠皮下移植瘤模型，运用RNA干扰等技术手段，基于自噬研究天冬多糖调控肿瘤血管生成、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作用，阐明其抵抗缺氧微环境应答机制，为提高中药TACE治疗肝癌的临床疗效提供实验依据。

目的：通过体外细胞实验及体内动物实验，探讨天冬多糖低氧条件下对肝癌细胞及肝癌移植瘤的作用，基于自噬探究天冬多糖下调HIF-1的机理。.方法：1.体外培养人肝癌细胞株Hep3B、HepG2，以培养基为空白对照阳性对照，用CKK法检测不同浓度的天冬多糖对人肝细胞株生长的影响，绘制量效曲线及生长曲线并计算IC50值；低氧条件下利用流式细胞技术、荧光染色、电镜、半定量RT-PCR和Western blot检测药物处理24h后的肝癌细胞自噬情况、自噬相关（LC3、Beclin-1）、HIF-1的表达。加入自噬及自噬相关（LC3、Beclin-1）基因敲减后肝癌细胞自噬及HIF-1的表达情况。.2. 体内动物实验中，构建裸鼠高转移性人肝癌细胞HCCLM3皮下移植瘤模型分别予瘤内注射介入给药，免疫组织化学法检测补体HIF-1等的表达。Western blot检测细胞自噬相关（LC3、Beclin-1）、HIF-1的表达。.结果：1.低氧条件下天冬多糖对人肝癌细胞株Hep3B、HepG2的生长的抑制作用随着浓度的增加(P＜0.05)；各种检测显示天冬多糖处理24h后的肝癌细胞抑制自噬、下调HIF-1表达水平。.2.实验裸鼠瘤体体积在瘤内注射药物天冬多糖组抑制肿瘤生长的作用明显；免疫组化及Western blot结果显示治疗组诱导肿瘤细胞凋亡及抑制肿瘤血管生成作用明显。.结论：天冬多糖在体外和体内均具有抑制的肝癌作用,上调caspase-3 、Bax,下调Bcl-2,下调HIF-1，其机制主要通过抑制自噬，抑制HIF-A的表达，抑制肿瘤血管生成，以及改善肝功能和提高免疫力的作用。