目的：构建过表达雷帕霉素靶蛋白（Mammalian Target of Rapamycin，mTOR）的人肝癌HepG2细胞，并研究中草药南蛇藤Celastrus orbiculatus Thunb.提取物对该细胞的影响。方法：利用分子生物学技术，将GV238-mTOR重组质粒转染人肝癌HepG2细胞，分别用荧光素酶活性检测和Western Blot实验，分析转染后的细胞中mTOR 的表达水平。人肝癌HepG2/mTOR⁺⁺细胞中，加入不同浓度的南蛇藤提取物（20、40、80、160、320μg·mL^-1）作用24h后，分析南蛇藤提取物对mTOR蛋白的作用。结果：经转染的人肝癌HepG2细胞中，mTOR蛋白的表达水平显著增加。南蛇藤提取物明显抑制肝癌细胞的增殖，并明显降低细胞内mTOR蛋白的表达。结论：本实验成功获得能稳定过表达mTOR的人肝癌HepG2细胞株。南蛇藤提取物明显抑制肝癌细胞内mTOR蛋白的表达水平。

This study aimed at investigating the effects of the extract of Chinese herb,Nansheteng(C.orbiculatus Thunb.),on human HepG2 cells through the overexpression of mTOR.The GV238-mTOR recombinant plasmids were transfected into HepG2 cells using molecular biological technology.The expression level of mTOR was evaluated by means of relative activity of luciferase and western blot.Human hepatic carcinoma HepG2/mTOR⁺⁺ cells were treated with C.orbiculatus extract in different concentrations (20,40,80,160 and 320μg·mL^-1) for 24h.The mTOR protein expression was detected by western blot.It was found that the protein expression of mTOR in transfected HepG2 cells was significantly enhanced.C.orbiculatus extract significantly inhibited the proliferation of HepG2/mTOR⁺⁺ cells.Simultaneously,C.orbiculatus extract inhibited mTOR at its protein level in a dose-dependent manner.In conclusion,we successfully constructed recombinant mTOR cloning vectors,and established the stable HepG2 cell line with the overexpression of mTOR.Besides,C.orbiculatus extract significantly inhibited mTOR protein expression in human hepatic carcinoma HepG2 cells.

国家自然科学基金委青年基金项目（81403232）：南蛇藤提取物靶向PI3K/Akt/mTOR信号通路抑制肝癌早期转移的作用及机制研究，负责人：钱亚云；国家自然科学基金委面上项目（81274141）：南蛇藤总萜通过协同抑制Notch和VEGF的表达阻断肝癌血管生成的作用机制，负责人：刘延庆；江苏省自然科学基金委面上项目（BK2012686）：南蛇藤提取物增强抑癌基因MASPIN表达抑制胃癌转移的机制研究，负责人：钱亚云；教育部博士点基金新教师类项目（20133250120003）：南蛇藤总萜靶向mTOR信号通路抑制肝癌转移的分子机制研究，负责人：钱亚云。