溶瘤腺病毒SG611联合顺铂对肝癌细胞HepG2的协同杀伤作用及其机制  

Synergistic cytotoxic effects and mechanism of oncolytic adenovirus SG611 combined with cisplatin on hepatocellular carcinoma Hep G2 cells

作　　者：胡朝霞[1,2] 台艳 刘炜 邱东波 张琪[2,3] 

机构地区：[1]中山大学附属第三医院感染科,广州3510630 [2]广东省肝脏疾病研究重点实验室,广州510630 [3]中山大学附属第三医院细胞与基因治疗转化医学研究中心,广州3510630

出　　处：《中华肝脏外科手术学电子杂志》2015年第2期52-56,共5页Chinese Journal of Hepatic Surgery(Electronic Edition)

基　　金：十二五重大科技专项(2012ZX10002016-023;2012ZX10002017-005;2012ZX10002010-001-007);国家自然科学基金(81170452;81370555);广东省自然科学基金(S20120011190);广东省科技计划项目(2012B061500005)

摘　　要：目的探讨溶瘤腺病毒SG611联合顺铂(DDP)对肝细胞癌(肝癌)细胞Hep G2的协同杀伤作用及其作用机制。方法利用携带GFP的腺病毒载体SG611感染人肝癌细胞Hep G2,流式细胞术检测SG611感染效率,CCK-8实验检测SG611联合DDP对肝癌细胞Hep G2杀伤效应,结晶紫染色法观察细胞毒性作用;4'6-二脒基-2-苯基吲哚(DAPI)染色法检测细胞凋亡率,Western blot检测E1A蛋白的表达。实验数据比较采用单因素方差分析和LSD-t检验。结果 SG611-EGFP感染肝癌细胞Hep G2荧光显微镜下观察,随着感染复数(MOI)的增加,GFP阳性细胞数目明显增加,流式细胞术检测SG611的感染效率分别为0.18%、6.36%、50.60%、73.20%、86.80%、90.50%,呈剂量依赖性。MOI=10的SG611与1.5μg/ml的DDP联合应用的细胞存活率为(33.2±1.2)%,明显低于单用SG611的(88.8±8.9)%(LSD-t=-7.83,P<0.05);且联合应用对肝癌细胞Hep G2的细胞毒性作用明显强于单用SG611。两者联合应用时肝癌细胞Hep G2凋亡率为(23.9±1.5)%,明显高于单用SG611、DDP的(15.3±1.0)%、(12.4±1.1)%(LSD-t=10.56,21.34;P<0.05);且联合应用时肝癌细胞Hep G2的E1A表达明显增强。结论溶瘤腺病毒SG611联合DDP对肝癌细胞Hep G2具有协同杀伤作用。SG611增加DDP化疗敏感性及DDP增强SG611增殖能力可能为其协同杀伤的作用机制。Objective To investigate the synergistic cytotoxic effects and the mechanism of oncolytic adenovirus SG611 combined with cisplatin（DDP） on hepatocellular carcinoma（HCC） Hep G2 cells. Methods Human HCC Hep G2 cells were infected by adenovirus vector SG611 carried with green fluorescent protein（GFP）. The infection efficiency of SG611 on Hep G2 cells were examined by flow cytometry. The synergistic cytotoxic effects of SG611 combined with DDP on Hep G2 cells were evaluated by cell counting rit（cck）-8 assay and the cytotoxicity was assessed by crystal violet staining. The 4＇,6-diamidino-2-phenylindole（DAPI） staining was used to detect the apoptosis. The expression of protein E1 A was examined by Western blot. The comparison of experimental data was conducted using one-way analysis of variance and LSD-t test. Results HCC Hep G2 cells infected by SG611-EGFP were observed under fluorescence microscope. The GFP positive cells increased apparently with the increasing multiplicity of infection（MOI）. The infection efficiency of SG611 detected by flow cytometry was 0.18%, 6.36%, 50.60%, 73.20%, 86.80% and 90.50%, which was with dose dependent. With the combined use of SG611（MOI =10） and 1.5 μg/ml DDP, the cell viability was（33.2±1.2）%, which was significantly lower than（88.8±8.9）% of single use of SG611（LSD-t=-7.83, P〈0.05）. The cytotoxic effects on HCC Hep G2 cells of combined use was significantly higher than that of single use of SG611. The apoptosis rate of HCC Hep G2 cells was（23.9±1.5）% for combined use, which was significantly higher than（15.3±1.0）,（12.4±1.1）% for single use of SG611 or DDP（LSD-t=10.56, 21.34; P〈0.05）. In addition, E1 A expression in HCC Hep G2 cells significantly increased when combined use. Conclusions Oncolytic adenovirus SG611 combined with DDP has synergistic cytotoxic effects on HCC Hep G2 cells. The action mechanism of the synergistic cytotoxic effects may be that the chemosensitivity of DDP is enhanced by SG611 and the prolife

关 键 词：癌 肝细胞 溶瘤腺病毒SG611 顺铂 药物协同作用 腺病毒E1A蛋白质类 

分 类 号：R735.7[医药卫生—肿瘤]