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 effect. CONCLUSION The telomerase inhihitor MST 312 has radio sensitizatizing effect on HepG2 cells, which may be related to enhancedradiation induced telomere damage and impaired DNA damage repair after radiation.
出处
《中华肿瘤防治杂志》
CAS
北大核心
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)
