焦健,王铁杰,王珏,付春旺,毕开顺,刘茜
JIAO Jian1,WANG Tie-jie2,WANG Jue2,FU Chun-wang3,BI Kai-shun1,LIU Qian1(1.School of Pharmacy

• 1:沈阳药科大学药学院
• 2:深圳市药品检验所
• 3:沈阳药科大学中药学院

摘要(Abstract)：

目的观察穿山龙提取物(extract from Dioscoreae nipponicae Rhizoma,DNR)联合哈尔满碱(harmane)对人肝癌细胞(HepG2)增殖抑制作用及诱导凋亡的机理。方法采用MTT法检测药物对细胞的生长抑制作用,应用等效曲线-相互指数法(isobole-interaction index method)评价两药的相互作用,倒置显微镜观察细胞形态学变化,流式细胞仪(FCM)检测细胞凋亡率,Western印迹法检测凋亡蛋白procaspase-3的表达。结果穿山龙提取物(DNR)与哈尔满碱质量浓度比为2∶1时,I(相互指数)=0.768<1,联合用药具有协同作用;质量浓度比为1∶2时,I=1.041≈1.0,联合用药具有相加作用。FCM法检测联合用药48、72 h细胞凋亡率与单独用药相比分别增加了10.44%、50.06%,差异具有统计学意义(P<0.05)。Western印迹法检测联合用药诱导HepG2细胞凋亡的机制之一可能与procaspase-3蛋白的表达有关。结论 DNR联合哈尔满碱在体外有明显的协同抗肿瘤的作用,其可能的机制是影响procaspase-3蛋白的表达。
Objective To study the synergistic antitumor effect and mechanism of harmane combined with extract from Dioscoreae nipponicae Rhizoma(DNR)on human hepatocellular carcinoma HepG2 cells.Methods The effect of DNR combined with harmane on the proliferation of HepG2 cells was measured by MTT reduction test.The isobole-interaction index method was applied to evaluate the synergy.Morphological changes of tumor cells were analyzed by inverted microscope.Cell apoptosis was detected by flow cytometry(FCM).Expression of procaspase-3 in the process of apoptosis was detected by Western blot.Results DNR combined with harmane showed synergistic effect when their ratio was 2∶ 1(I<1).After cells were treated with drugs for 48 h and 72 h,cell apoptotic ratio of the mixtures was 10.44%,50.06%more than DNR alone.The mixtures of cell apoptotic ratio was different between DNR and harmane alone(P<0.05).The mechanism of action could have effect on procaspase-3 expression.Conclusions DNR and harmane have synergistic anticancer effect on HepG2 cells,and the mechanism maybe related with procaspase-3 expression.

关键词(KeyWords)： 穿山龙;哈尔满碱;人肝癌细胞;协同作用;细胞凋亡;半胱天冬酶-3酶原
Dioscoreae nipponicae Rhizoma;harmane;HepG2;synergy;apoptosis;procaspase-3

Abstract:

Keywords:

基金项目(Foundation): 广东省科技计划资助项目(2010A030100018)

作者(Author): 焦健,王铁杰,王珏,付春旺,毕开顺,刘茜
JIAO Jian1,WANG Tie-jie2,WANG Jue2,FU Chun-wang3,BI Kai-shun1,LIU Qian1(1.School of Pharmacy

DOI: 10.14066/j.cnki.cn21-1349/r.2012.10.009

参考文献(References)：

• [1]刘江涛,陈信,王玉芝.穿山龙粗提物抗肿瘤体外实验研究[J].中国中医药信息杂志,2004,11(3):206-207.
• [2]WANG Tie-jie,LIU Zhong-bo,LI Jun,et al.Determina-tion of protodioscin in rat plasma by liquid chromatogra-phy-tandem mass spectrometry[J].Chromatography B,2007,848(2):363-368.
• [3]王艳.刺蒺藜药理作用及化学成分的研究概况[J].北京中医学院学报,1989,12(6):30-32.
• [4]常新全,丁丽霞.中药活性成分手册[M].北京:学苑出版社,2002:2235.
• [5]WANG Jue,ZU Xu-yu,JIANG Yu-yang.Five furostanolsaponins from fruits of Tribulus Terrestris and their cyto-toxic activities[J].Natural Product Research,2011,23(15):1436-1444.
• [6]徐端正.药物联合作用评价[J].生理科学进展,1992,23(3):248-253.
• [7]MING Zhi-jun,HU Yong,QIU Yu-hua,et al.Synergisticeffects ofβ-aescin and 5-fluorouracil in human hepato-cellular carcinoma SMMC-7721 cells[J].Phytomedi-cine,2010,17:57.
• [8]李海军,王辉,方全华,等.康莱特注射液诱导肝癌细胞凋亡及其对凋亡蛋白procaspase-3和caspase-9表达的影响[J].肿瘤,2010,30(9):740-743.