斑蝥酸钠联合化疗药对肝癌的协同增效及其机制研究  

Study on Synergistic Effect of Sodium Cantharidinate Combined with Chemotherapeutic Drugs on Hepatic Carcinoma and Its Effective Mechanism

作　　者：赵若琳[1] 陈美娟[1] 赵凤鸣[1] 许冬青[1] 张旭[1] 周坤福[1] 

机构地区：[1]南京中医药大学,江苏南京210023

出　　处：《中药材》2014年第11期1938-1946,共9页Journal of Chinese Medicinal Materials

基　　金：江苏省高校优势学科建设工程项目;江苏省自然科学基金(BK20131415)

摘　　要：目的:研究斑蝥酸钠(Sodium Cantharidinate,SCA)联合氟尿嘧啶(Fluorouracil,5-FU)或顺铂(Cisplatin,DDP)对肝癌细胞(SMMC-7721)协同增效和减少对正常肝细胞(LO-2)杀伤的作用及其相关机理。方法:采用MTT法筛选具有减毒增效作用的最佳药物配比;流式细胞术分析该浓度配比的药物对细胞凋亡率的影响;Hoechst-33258荧光染色法观察凋亡细胞核形态的变化;Western blot分析该浓度配比的药物对SMMC-7721和LO-2细胞中凋亡和生长抑制相关蛋白的表达调控。结果:MTT检测结果显示,SCA、5-FU、DDP分别单独使用时对SMMC-7721的细胞增殖抑制均呈时间和剂量依赖关系。SCA在12、24 h分别对LO-2与SMMC-7721抑制具有显著性作用(P<0.01),其对LO-2抑制作用较小。并且,SCA和DDP以2.5、2μg/m L的浓度配比处理细胞48 h时,相对于其他处理组,其对SMMC-7721抑制率较高,对LO-2抑制率相对较低,故确定该浓度配比为最佳浓度配比;流式细胞术检测结果显示,2.5μg/m L SCA与2μg/m L DDP联合作用相对于SCA(2.5μg/m L)或DDP(2μg/m L)单独作用,其对SMMC-7721凋亡诱导作用有显著性差异(P<0.01)。倒置显微镜观察及Hoechst-33258荧光染色观察均发现,在该药物配比浓度下作用48 h后细胞形态出现显著的凋亡特征;Western-blot结果显示,该药物配比能显著上调SMMC-7721细胞中BAX、P53和P21蛋白的表达,并下调BCL-2、Casepase-3、p-Erk、p-Ras和p-c-Raf蛋白的表达。同时,其对LO-2相关蛋白的表达影响较小。结论:以上结果显示在2.5μg/m L SCA+2μg/m L DDP的配比浓度下,药物显示出对肝癌细胞较好的抑制作用及对正常肝细胞相对较低的杀伤作用。其抗癌机制主要和抑制Erk信号通路及通过线粒体途径诱导凋亡有关。Objective： To study the synergistic effect on hepatoma cell（ SMMC-7721） and the reduction killing effect on normal liver cells（ LO-2） treated with sodium cantharidinate（ SCA） in combination with fluorouracil（ 5-FU） or cisplatin（ DDP） as well as the related mechanism. Methods： MTT assay was used to select the best ratio of SCA with 5-FU or SCA with DDP which had less toxicity on LO-2 cell line and had synergistic effect on SMMC-7721 cell line; Flow cytometry assay was used to analyze the apoptosis-induction of the different ratio of drugs on both cell lines; Hoechst-33258 fluorescent staining assay was used to observe the nuclear morphological changes of cells; Immunoblotting assay was used to analyze the Ras / Raf / ERK1 /2 signaling pathway and the apoptosis related signaling pathway in both cell lines. Results： MTT assay indicated that the proliferation inhibition of SCA,5-FU and DDP on SMMC-7721 cell line was in a time-and dose-dependent manner respectively. Among them,SCA had a more significant inhibition on SMMC-7721 cell line than on LO-2 after 12 h or 24 h treatment（ P〈0. 01）. Moreover,after a treatment of 48 h,the ratio of 2. 5 μg / m L SCA and 2 μg / m L DDP showed a more significant inhibition on SMMC-7721 cell line than on LO-2 cell line,which was then be considered as the optimal concentration ratio for the following experiment. Co-treatment of SCA（ 2. 5 μg / m L） with DDP（ 2 μg / m L） induced a more significant apoptosis on SMMC-7721 cell line compared with single treatment with SCA（ 2. 5 μg / m L） or DDP（ 2 μg / m L） respectively（ P〈0. 01）. After a 48 h treatment of the optimal ratio of drugs,the significant morphological apoptotic characteristics were observed both under inverted microscope and by Hoechst-33258 fluorescent staining assay in both cell lines. The results of Western blot assay showed that this ratio of drugs could significantly increase the protein expression of Bax,P53 and P21 and decreased the expression of BCL-2,Casepase-3

关 键 词：斑蝥酸钠 顺铂 氟尿嘧啶 肝癌 凋亡 

分 类 号：R285.5[医药卫生—中药学]