基于“谱-效关系”的藏药雪灵芝抑制人肝癌细胞HepG-2细胞增殖的物质基础及机制研究

Hepatocellular Carcinoma (HCC) remains the third leading cause of cancer-related mortality globally. Half of the diagnosed liver cancer cases and deaths were estimated to occur in China. As known, surgical resection, embolization, ablation, and chemotherapy play important roles in the treatment of HCC,but it is limited to a significant extent by its toxicities,significant resistance to available chemotherapeutic agents,side effects and complexities. Given the poor prognosis associated with HCC and limited treatment options outside of surgery, patients seek additional therapies to improve quality of life or survival.Our research group first found that Tibetan medicine Arenaria kansuensis extraction can apparently inhibit proliferation of human hepatocellular carcinoma Hep G-2 cells which of pharmacodynamic material basis is unkown.This project intend to analyze and reveal effective chemical compounds or effective compounds group of Arenaria kansuensis suppressed Hep G-2 cells growth with the guidance of spectrum-effect relationship on the basis of former research. And the correlation between chromatographic fingerprint and anti-proliferation -treating function of effective fractions will be identified using grey relational analysis. Fundamental research methods are chromatographic separations and activities screening in vitro. Lead compound or effective compounds group suppressed proliferation of Hep G-2 cells is expected to be discovered by the research group. At the same time, we will investigate their influences on mRNA and protein expressions of Bcl-2, Bax and Cycling D1 related with proliferation and apoptosis of Hep G-2 cells, which aims to take the first steps in approaching the action mechanism of effective chemical compounds or compounds group of Arenaria kansuensis. Development of this project is expected to provide the theoretical and experimental basis for revealing the pharmacodynamic material basis of the traditional Tibetan medicine Arenaria kansuensis for the treatment of HCC which is a new function.

肝细胞癌（HCC）是世界第三大恶性肿瘤，以我国发病率最高，严重威胁人类健康。现有化疗药物的毒副作用与耐药显著影响治疗效果和预后。本项目在前期系统的化学成分研究的基础上，首次发现藏药雪灵芝乙醇提取物的乙酸乙酯萃取物对HepG-2细胞具有显著的增殖抑制作用，但药效物质基础尚不明确。本项目拟以此有效部位群为研究对象，采用色谱分析与细胞筛选相结合的手段，在基于灰色关联分析的“谱-效关系”指导下，从前期发现的有效部位群中寻找雪灵芝抑制HepG-2细胞增殖、促进凋亡的活性成分或成分群；并通过观察活性成分或成分群对HepG-2细胞增殖与凋亡的相关基因（Bcl-2、Bax、Cycling D1）转录表达的影响，初步探讨其抑制增殖可能的作用途径。为阐明传统藏药雪灵芝抑制HepG-2细胞增殖的物质基础提供理论和实验依据，为今后开发雪灵芝防治肝癌的药理作用奠定研究基础。

本项目前期研究首次发现藏药雪灵芝乙醇提取物的乙酸乙酯萃取物对HepG-2细胞具有显著的增殖抑制作用。本项目以此有效部位群为研究对象，采用色谱分析与细胞筛选相结合的手段，在基于灰色关联分析的“谱-效关系”指导下，从前期发现的有效部位群中定位了雪灵芝抑制HepG-2细胞增殖、促进凋亡的两个活性子部位，并且从中分离得到一个五环三萜皂苷群，且本次研究已经鉴定了其中含量较高的3个单体皂苷化合物的结构；Hoechst 33258染色结果表明，筛选出的两个活性子部位具有一定的促进HepG-2细胞凋亡的作用；而Annexin V/PI 双染流式细胞术检测结果也进一步证实了这两个活性子部位对HepG-2细胞的增殖抑制及凋亡诱导作用，且呈现剂量依赖性；Western Blot实验初步探索了其作用机制可能与调控HepG-2细胞增殖及凋亡相关基因（Bc1-2、Bax、Cyclin D1）的表达有关。本研究为阐明传统藏药雪灵芝抑制HepG-2细胞增殖的物质基础提供理论和实验依据，为今后开发雪灵芝防治肝癌的药理作用奠定研究基础。项目资助发表论文3篇，正在培养硕士生2名。剩余经费计划用于本项目后续研究的支出。