MST-312对人肝癌HepG2细胞辐射敏感性影响研究  

Effects of telomerase inhibitor MST-312 on human hepatoma HepG2 cells radiosensitivity

作　　者：王亚丽[1,2] 孙超[2] 张昕[2] 谢漪[2] 杨立娜[3] 刘圆圆[2] 赵邱越[2] 甘露[2] 张红[2] 

机构地区：[1]兰州大学药学院,甘肃兰州730000 [2]中国科学院近代物理研究所,甘肃兰州730000 [3]兰州大学生命科学院,甘肃兰州730000

出　　处：《中华肿瘤防治杂志》2015年第1期1-6,共6页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家重点基础研究发展计划(973计划;2010CB834202);甘肃省重大科学技术专项(0702NKDA045;0806RJYA020);中国科学院西部之光人才培养计划(XB106012)

摘　　要：目的研究端粒酶抑制剂MST-312对人肝癌细胞HepG2辐射敏感性影响。方法 MTT实验检测0、2、4、6、8和10μmol/L的MST-312对HepG2细胞的增殖抑制作用;采用克隆形成实验检测HepG2细胞存活率,观察MST-312对细胞辐射增敏性的影响;流式细胞术分析细胞周期及凋亡率;Hoechst 33258荧光染色法检测细胞凋亡的形态变化;蛋白质印迹法检测γ-H2AX蛋白表达水平,衡量DNA损伤的程度。结果 MTT法检测结果显示,MST-312对HepG2细胞增殖抑制作用呈剂量依赖性,4μmol/L MST-312对HepG2细胞的增殖抑制率<10%,而且对细胞的周期分布没有明显影响。克隆形成实验结果显示,MST-312预处理2h后再给予X射线辐照的联合组克隆形成率为(17.68±4.01)%,较辐照组(62.60±7.91)%明显降低,F=9.773,P=0.014。流式细胞术分析结果表明,随着时间的延长,联合组G2/M期的比例由(20.56±0.75)%下降至(5.82±0.45)%,而Sub-G1峰值由(6.13±0.43)%上升至(15.78±0.89)%,即随着G2/M期阻滞比例下降的同时Sub-G1峰值比例在上升。Hoechst 33258荧光染色结果表明,在X射线处理后12h,联合组比辐照组出现了更加明显的细胞凋亡形态变化。蛋白质印迹法检测结果显示,在辐照后24h,联合组γ-H2AX蛋白表达量(614.20±21.13)%,高于辐照组(421.78±19.11)%,F=14.513,P=0.005。提示DNA损伤严重,未完全修复。结论端粒酶抑制剂MST-312促进了辐射诱导的细胞凋亡,对HepG2细胞具有辐射增敏作用,其机制可能与加剧辐射诱导的端粒损伤和抑制辐射后DNA损伤修复有关。OBJECTIVE This study aims to test radiation-sensitivitizing effect induced by telomerase inhibitor MST-312 on human hepatoma cellline HepG2. METHODS The cytotoxieity of MST-312 on HepG2 cells was tested by MTT assay at a concentration of 0,2,4,6,8,10μmol/L respectively. The clonogenie assay was performed to evaluate the cell surviving fractions and determine the radiation-sensitizing effect of MST-312 on HepG2 cells. The cell cycle distribu- tion and apoptosis after radiation were analyzed by flow cytometry analysis. Apoptosis paralleled morphological changes were detected with Hoechst 33258 fluorescent staining. As a mark of DNA damage, y H2AX protein expression was measured by Western Blot. RESULTS MTT assay showed that MST-312 decreased the proliferation of HepG2 cells in a dose-dependent manner. Four 〉mol/L of MST-312 inhibited proliferation of HepG2 cells less than 10% during the test period, and had no significant effect on the cell cycle distribution. The radio-sensitivity of HepG2 cells was examined by elonogenie assay, which revealed that colony-forming efficiency of the MST-312 pretreatment followed by X-rays was （17.68± 4. 01 ）%, F = 9. 773, P = 0. 014, which was obviously lower compared with irradiation group [-（ 62. 60 ± 7.91）%]. Flow eytometry analysis showed greater increases in apoptotic cells after the combined treatment, and G2/M phase fraction decreased from （20.56±0.75）% to （5.82±0.45）% with time, simultaneously the proportion of Sub-G1 increased from （6.13±0.43）% to （15.78±0.89）%. This study further confirmed the apoptosis fraction of HepG2 cell after X-ray radiation with Hoechst33258 staining, and lhe results showed higher level of cell apoptosis in the combined treatment group. The Western Blot results indicated a prolonged higher expression level of ）＇ H2AX in samples from corn bined treament [（614. 20±21. 13）%,F= 14. 513, P=0. 005], which was higher compared with irradiation group [（421.78±19. 11）%] and which implied an enhanced DNA damaging

关 键 词：肝肿瘤 HEPG2细胞 端粒酶抑制剂 X-射线 辐射增敏 流式细胞术 

分 类 号：R735.7[医药卫生—肿瘤]