上皮间质转换（EMT）是原发性肝癌早期侵袭转移的重要机制，PI3K/Akt/mTOR信号通路在EMT中起关键作用。前期免疫组化技术分析48例临床肝癌标本后证实肝癌转移与EMT密切相关，并发现南蛇藤总萜（发明专利号ZL200710025343.3）明显抑制肝癌HepG2细胞中mTOR蛋白的表达水平。本研究首先进一步研究临床标本，分析mTOR与肝癌EMT的相关性；其次，建立mTOR高表达/RNAi沉默表达的肝癌细胞模型，研究南蛇藤总萜通过PI3K/Akt/mTOR信号通路抑制肝癌细胞EMT的分子机制。最后，联合裸鼠和Tg (fli1a: EGFP) y1斑马鱼两种肝癌转移模型，用体内分子示踪技术，直接观察南蛇藤总萜对肝癌移植瘤及微转移病灶的影响。本项目可阐明南蛇藤总萜抑制肝癌早期转移的分子机制，为开发具有自主知识产权的抗癌新药奠定基础。

Epithelial–mesenchymal transition (EMT) is the key molecular mechanisms responsible for HCC development and early metastasis.And the PI3K/Akt/mTOR signaling pathway has been known to play a critical role in the EMT. Immunohistochemistry was used in 48 patients with hepatocellular carcinoma（HCC） to confirm that HCC metastasis is associated with EMT. And we found that the Celastrus Orbiculatus extracts (the patent of invention No. ZL200710025343.3) significantly inhibited mTOR expression in HepG2 cells. On this basis, the surgical specimens with hepatocellular carcinoma will be further studied. Then, establishment overexpression /RNAi knockdown of mTOR in HepG2 cells to investigate molecular mechanisms underlying the Celastrus Orbiculatus extracts inhibit EMT by targeting PI3K/Akt/mTOR signaling pathways. In vivo, we will combine nude mice and EGFP tag Tg (fli1a: EGFP) y1 zebrafish hepatocellular carcinoma metastasis model, to observe the affections of he Celastrus Orbiculatus extracts on the tumor microenvironment by using molecular tracer technique. This study will reveal the molecular mechanism of early metastasis HCC, and exploit a new anticancer drug with independent intellectual property.

为了揭示南蛇藤提取物抑制肝癌转移的分子机制，本课题结合了体内外实验进行研究。首先，我们用氯化钴体外诱导法建立肝癌HepG2细胞的缺氧微环境，加入不同浓度的COE作用24 h后发现，缺氧微环境下，COE抑制人肝癌HepG2细胞的EMT过程，其分子机制可能与HIF-1α/Twist1信号通路和PI3k/Akt/mTOR信号通路相关。接着，我们用分子生物学的方法分别建立起稳定的高表达mTOR 的HepG2/mTOR+细胞模型，以及敲除mTOR 表达的HepG2/mTOR-细胞模型。加入不同浓度的COE作用24 h，采用实时荧光定量PCR、Western Blot等技术，研究南蛇藤总萜对EMT相关信号通路的影响。结果表明南蛇藤提取物促进HepG2/mTOR+人肝癌细胞以及HepG2/mTOR-人肝癌细胞的凋亡，明显抑制HepG2/mTOR+细胞及HepG2 / mTOR-细胞内的mTOR相关信号通路的表达水平。同时，联合利用斑马鱼和裸鼠两种动物模型进行体内研究。斑马鱼胚胎，于受精后48h开始给药，结果发现，与对照组相比，南蛇藤提取物各浓度组明显增加E-cadherin 的表达量，降低vimentin和mTOR信号通路相关蛋白的表达水平。HepG2/mTOR-人肝癌细胞注射到裸鼠皮下，成瘤后，用不同浓度的南蛇藤提取物灌胃28天，发现南蛇藤提取物抑制肝癌的生长，并且促进肿瘤细胞的凋亡。本研究表明南蛇藤提取物能在一定程度上逆转人肝癌细胞EMT，其分子机制可能与mTOR信号通路相关，mTOR可作为临床治疗肝癌的新靶点。