贵州医科大学学报

2020, v.45;No.239(08) 882-886+927

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乐伐替尼诱导肝癌耐药细胞株的构建及其生物学特性探析
Establishment and Biological Characteristics of Levatinib-Resistant Hepatocellular Carcinoma Cell Line

杨哲豪;喻超;潘耀振;邓路;郑迪杰;孙诚谊;
YANG Zhehao;YU Chao;PAN Yaozhen;DENG Lu;ZHENG Dijie;SUN Chengyi;Department of Hepatobiliary Surgery,the Affiliated Hospital of Guizhou Medical University Key Laboratory of Liver,Gallbladder,Pancreas and Spleen,Guizhou Medical University Institute of Liver,Gallbladder,Pancreas and Spleen Diseases Diseases of Guizhou;

摘要(Abstract):

目的:构建乐伐替尼诱导的原发性肝细胞癌(HCC) SMMC-7721耐药细胞株,并探讨其生物学特性。方法:选取HCC细胞株SMMC-7721进行培养传代,采用药物逐步递增法建立体外乐伐替尼诱导的SMMC-7721耐药细胞株(耐药组),以未经处理的肝癌细胞为对照组;采用CCK-8法检测乐伐替尼、阿霉素、氟尿嘧啶和顺铂对两组细胞的半数抑制浓度(IC50),并计算乐伐替尼耐药指数(RI);采用Real time-PCR检测两组细胞耐药基因MDR1、LRP、MRP2、GST-pi、TopoⅡɑ和BCRP的表达,采用Western blot检测两组细胞P-gp、LRP、MRP2、GST-pi、TopoⅡɑ和BCRP蛋白的表达。结果:乐伐替尼对对照组和耐药组SMMC-7721细胞IC50分别为(0.15±0.03)μmol/L和(4.34±0.12)μmol/L,RI为28;阿霉素、氟尿嘧啶和顺铂对耐药组SMMC-7721细胞的IC50较对照组降低(P <0.05);耐药组SMMC-7721细胞中MDR1、LRP、MRP2、GST-pi、TopoⅡɑ及BCRP耐药基因的相对表达量均较对照组下降(P <0.05或P <0.01),耐药组SMMC-7721细胞中P-gp、LRP、MRP2、GST-pi、TopoⅡɑ及BCRP蛋白表达均较对照组降低(P <0.05)。结论:实验成功诱导获得耐乐伐替尼的体外肝癌SMMC-7721细胞株,该细胞株对阿霉素、氟尿嘧啶和顺铂的敏感性增高,其体制可能与耐药基因MDR1、LRP、MRP2、GST-pi、TopoⅡɑ及BCRP表达降低有关。
Objective: To construct levatinib-induced hepatocellular carcinoma( HCC) SMMC-7721 drug-resistant cell line and explore its biological characteristics. Methods: HCC cell line SMMC-7721 was selected to culture and subculture. The levatinib-resistant cell line was established by step-by-step drug-increasing method. The untreated HCC cells were selected as the control group and the levatinibresistant cells as the drug-resistant group. The median inhibition concentration( IC_(50)) of levatinib,doxorubicin,fluorouracil and cisplatin of both groups was detected by CCK-8 assay and the drug resistance index( RI) of levatinib was calculated. Real time-PCR was used to detect the expression of drug-resistance genes MDR1,LRP,MRP2,GST-pi,TopoⅡɑ and BCRP in two groups. Western blot was used to detect the expression of P-gp,LRP,MRP2,GST-pi,TopoⅡɑ and BCRP proteins of the cells. Results: The IC50 of levatinib on SMMC-7721 cells of two groups were( 0. 15 ± 0. 03) μmol/L and( 4. 34 ± 0. 12) μmol/L( P < 0. 05). The drug resistance index was 28,and the IC50 of doxorubicin,fluorouracil and cisplatin of the drug-resistant group was lower than that in the control group( P < 0. 05). The expression of genes MDR1,MRP2,LRP,GST-pi,TopoⅡɑ and BCRP was low in drug-resistant cells( P < 0. 05 or P < 0. 01) while the expression of protein P-gp,LRP,MRP2,GST-pi,Topo Ⅱ ɑ and BCRP decreased in drug-resistant cells. Conclusion: The experiment successfully established HCC cell line SMMC-7721 with levatinib-resistance and found that the increased sensitivity to doxorubicin,fluorouracil and cisplatin may be related to the low expression of genes MDR1,LRP,MRP2,GST-pi,TopⅡɑ and BCRP in levatinib-resistance HCC cells.

关键词(KeyWords): 癌,肝细胞;分子靶向治疗;乐伐替尼;耐药细胞;化疗敏感性;多药耐药
carcinoma,hepatocellular;molecular yargeted therapy;levatinib;drug-resistant cells;chemosensitivity;multidrug resistance

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(81860505,81860506);; 贵州省科学技术厅-贵州医科大学附属医院联合基金[黔科合LH字(2016)7229];贵州省肝胆外科临床医学研究中心[黔科合平台人才(2017)5404]

作者(Author): 杨哲豪;喻超;潘耀振;邓路;郑迪杰;孙诚谊;
YANG Zhehao;YU Chao;PAN Yaozhen;DENG Lu;ZHENG Dijie;SUN Chengyi;Department of Hepatobiliary Surgery,the Affiliated Hospital of Guizhou Medical University Key Laboratory of Liver,Gallbladder,Pancreas and Spleen,Guizhou Medical University Institute of Liver,Gallbladder,Pancreas and Spleen Diseases Diseases of Guizhou;

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DOI: 10.19367/j.cnki.2096-8388.2020.08.003

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