双硫仑联合铜离子对乐伐替尼耐药肝癌细胞Huh7增殖及凋亡的影响  

Effects of disulfiram combined with copper ions on proliferation and apoptosis of lenvatinib resistance Huh7 cells

作　　者：董博文[1] 李子一 李伟东[2] 代文杰[3] DONG Bowen;LI Ziyi;LI Weidong;DAI Wenjie(Key Laboratory of The Ministry of Education of Hepatosplenic Surgery,The First Affiliated Hospital of Harbin Medical University,Harbin 150001,P.R.China;The Third Department of General Surgery,The Fourth Affiliated Hospital of Harbin Medical University,Harbin 150001,P.R.China;Department of Thyroid Surgery,The First Affiliated Hospital of Harbin Medical University,Harbin 150001,P.R.China)

机构地区：[1]哈尔滨医科大学附属第一医院肝脾外科教育部重点实验室,哈尔滨150001 [2]哈尔滨医科大学附属第四医院普外三科,哈尔滨150001 [3]哈尔滨医科大学附属第一医院甲状腺外科,哈尔滨150001

出　　处：《中国普外基础与临床杂志》2020年第2期168-172,共5页Chinese Journal of Bases and Clinics In General Surgery

摘　　要：目的建立乐伐替尼肝细胞肝癌(简称"肝癌")细胞耐药模型,以研究双硫仑联合铜离子对乐伐替尼耐药细胞Huh7增殖和凋亡的影响。方法通过CCK8法检测乐伐替尼作用下Huh7细胞的半抑制浓度(IC50),采用浓度递增法诱导Huh7细胞乐伐替尼耐药。各组细胞处理:对照组为Huh7细胞加普通培养液培养后的细胞;敏感组为Huh7细胞加10μmol/L乐伐替尼培养后的细胞;耐药组为耐药Huh7细胞加10μmol/L乐伐替尼培养后的细胞;联合组为耐药Huh7细胞加10μmol/L乐伐替尼+1μmol/L双硫仑+0.5μmol/L铜离子培养后的细胞;抑制剂组为耐药Huh7细胞加10μmol/L乐伐替尼+10μmol/L Akt抑制剂MK2206培养后的细胞。CCK8法及流式细胞术分别检测细胞的存活率和凋亡率;Western blot法检测磷酸化-Akt(p-Akt)和半胱氨酸蛋白酶9(caspase-9)和B淋巴细胞瘤2(Bcl-2)蛋白表达;Transwell法检测细胞侵袭性。结果乐伐替尼耐药细胞系成功建立,选取10、20、40、60、80及160μmol/L 6个乐伐替尼浓度对Huh7细胞作用48 h后计算得出细胞IC50为12.35μmol/L。耐药组细胞存活率明显高于敏感组(P<0.01),联合组细胞存活率明显低于耐药组(P<0.01),抑制剂组细胞存活率明显低于耐药组(P<0.05)。与耐药组比较,联合组及抑制剂组p-Akt蛋白表达明显降低(P<0.01)、caspase-9明显升高(P<0.01),而Bcl-2蛋白在耐药组和联合组间比较差异无统计学意义(P>0.05)。联合组细胞凋亡率明显高于耐药组(P<0.01)。Transwell结果显示联合组的肿瘤细胞侵袭数明显少于耐药组(P<0.01)。结论双硫仑联合铜离子能增加乐伐替尼对乐伐替尼耐药肝癌细胞Huh7的敏感性,同耐药组相比其细胞抑制率明显提高,其机制可能与抑制磷脂酰肌醇-3-激酶/Akt通路及促进caspase-9蛋白表达有关。Obejective To establish the lenvatinib-resistant model of hepatocellular carcinoma(HCC) in vitro in order to study the effect of disulfiram combined with copper ions on the proliferation and apoptosis of lenvatinib-resistant Huh7 cells. Methods The half-inhibitory concentration(IC50) of Huh7 cells treated with lenvatinib was detected by the CCK8 method, and the resistance of Huh7 cells to lenvatinib was induced by increasing concentration method. The cell treatment of each group: The Huh7 cells cultured with common culture medium and with 10 μmol/L lenvatinb in the control group and lenvatinib sensitive group, respectively;The lenvatinib-resistant Huh7 cells cultured with 10 μmol/L lenvatinb, with 10 μmol/L lenvatinib+1 μmol/L disulfiram+0.5 μmol/L copper ions, and with 10 μmol/L lenvatinib+10 μmol/L Akt inhibitor MK2206 in the lenvatinib-resistant group, combined drugs group, and inhibitor group, respectively.The cell survival and apoptosis rates were detected by the CCK8 and flow cytometry, respectively. The Western blot method was used to detect the expressions of phospho-protein kinase B(p-Akt), cysteine aspartic acid specific protease(caspase-9), and B-cell lymphoma-2(Bcl-2) proteins. The Transwell assay was used to detect the cell invasiveness.Results The lenvatinib-resistant cell line was successfully established. The concentrations of 10, 20, 40, 60, 80 and160 μmol/L leventinib were applied to the Huh7 cells for 48 h, and the IC50 was calculated to be 12.35 μmol/L. The cell survival rate of the lenvatinib-resistant group was significantly higher than that of the lenvatinib sensitive group(P<0.01),which in the combined drugs group was significantly lower than that of the lenvatinib-resistant group(P<0.01), which in the inhibitor group was significantly lower than that of the lenvatinib-resistant group(P<0.05). The apoptosis rate of the combined drugs group was significantly higher than that of the lenvatinib-resistant group(P<0.01). Compared with the lenvatinib-resistant group, the expression of p

关 键 词：肝细胞肝癌 双硫仑铜离子 乐伐替尼耐药 磷酸化蛋白激酶B 半胱氨酸蛋白酶9 

分 类 号：R735.7[医药卫生—肿瘤]