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

doi:10.3877/cma.j.issn.2095-3232.2015.02.014

中华肝脏外科手术学电子杂志 ›› 2015, Vol. 04 ›› Issue (02) : 119 -124. doi: 10.3877/cma.j.issn.2095-3232.2015.02.014

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基础研究

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

胡朝霞¹, 台艳², 刘炜², 邱东波³, 张琪⁴^,^†(

)

^1. 510630　广州，中山大学附属第三医院感染科；510630　广州，广东省肝脏疾病研究重点实验室
^2. 510630　广州，广东省肝脏疾病研究重点实验室
^3. 510630　广州，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
^4. 510630　广州，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；510630　广州，广东省肝脏疾病研究重点实验室

收稿日期:2015-02-03 出版日期:2015-04-10
通信作者: 张琪
基金资助:
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)

Synergistic cytotoxic effects and mechanism of oncolytic adenovirus SG611 combined with cisplatin on hepatocellular carcinoma HepG2 cells

Zhaoxia Hu¹, Yan Tai², Wei Liu², Dongbo Qiu³, Qi Zhang⁴^,^†()

^1. Department of Infectious Disease, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China; Guangdong Provincial Key Laboratory of Liver Disease, Guangzhou 510630, China
^2. Guangdong Provincial Key Laboratory of Liver Disease, Guangzhou 510630, China
^3. Cell and Gene Therapy Translational Medicine Research Center, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China
^4. Guangdong Provincial Key Laboratory of Liver Disease, Guangzhou 510630, China; Cell and Gene Therapy Translational Medicine Research Center, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China

Received:2015-02-03 Published:2015-04-10
Corresponding author: Qi Zhang
About author:
Corresponding author: Zhang Qi, Email:zhangq27@mail.sysu.edu.cn

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引用本文：

胡朝霞, 台艳, 刘炜, 邱东波, 张琪. 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[J]. 中华肝脏外科手术学电子杂志, 2015, 04(02): 119-124.

Zhaoxia Hu, Yan Tai, Wei Liu, Dongbo Qiu, Qi Zhang. Synergistic cytotoxic effects and mechanism of oncolytic adenovirus SG611 combined with cisplatin on hepatocellular carcinoma HepG2 cells[J]. Chinese Journal of Hepatic Surgery(Electronic Edition), 2015, 04(02): 119-124.

目的

探讨溶瘤腺病毒SG611联合顺铂（DDP）对肝细胞癌（肝癌）细胞HepG2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HepG2，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HepG2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HepG2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1.5 μg/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HepG2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HepG2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HepG2的E1A表达明显增强。

结论

溶瘤腺病毒SG611联合DDP对肝癌细胞HepG2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。

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

Objective

To investigate the synergistic cytotoxic effects and the mechanism of oncolytic adenovirus SG611 combined with cisplatin (DDP) on hepatocellular carcinoma (HCC) HepG2 cells.

Methods

Human HCC HepG2 cells were infected by adenovirus vector SG611 carried with green fluorescent protein (GFP). The infection efficiency of SG611 on HepG2 cells were examined by flow cytometry. The synergistic cytotoxic effects of SG611 combined with DDP on HepG2 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 E1A was examined by Western blot. The comparison of experimental data was conducted using one-way analysis of variance and LSD-t test.

Results

HCC HepG2 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 HepG2 cells of combined use was significantly higher than that of single use of SG611. The apoptosis rate of HCC HepG2 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, E1A expression in HCC HepG2 cells significantly increased when combined use.

Conclusions

Oncolytic adenovirus SG611 combined with DDP has synergistic cytotoxic effects on HCC HepG2 cells. The action mechanism of the synergistic cytotoxic effects may be that the chemosensitivity of DDP is enhanced by SG611 and the proliferation of SG611 is enhanced by DDP.

Key words: Carcinoma, hepatocellular, Oncolytic adenovirus SG611, Cisplatin, Drug synergism, Adenovirus E1A proteins

图1 溶瘤腺病毒SG611感染肝癌细胞HepG2镜下观察（荧光显微镜　×200）

图2 结晶紫染色法检测SG611联合DDP对肝癌细胞HepG2的细胞毒性作用

图3 DIPA染色检测肝癌细胞HepG2凋亡情况

图4 Western blot检测E1A蛋白表达

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