【作者】 王树彬；

【导师】 孙伟建； 周平坤；

【作者基本信息】 中国人民解放军军事医学科学院 ， 肿瘤学， 2010， 硕士

【摘要】 一、目的观察香兰素衍生物6-溴异香兰素(6-溴-5-羟基-4-甲氧基苯甲醛,BVAN08)对人脑胶质瘤U-251细胞增殖的影响,及其放射增敏作用和相关机制。为开发其为新的抗肿瘤药物提供进一步理论和实验依据。二、方法采用MTT法检测不同浓度BVAN08对U-251细胞的细胞毒性作用；通过克隆形成率法测定U251细胞在不同剂量60Coγ射线照射下的存活曲线,检测和比较照射前和照射后加入BVAN08对细胞放射敏感性的影响；流式细胞术检测U-251细胞经BVAN08作用后细胞周期及凋亡的变化；光学显微镜观察BVAN08作用后细胞形态学变化；透射电镜检测细胞自吞噬死亡；Western blot检测BVAN08作用后DNA修复蛋白DNA-PKcs表达变化。三、结果MTT检测结果表明BVAN08对U-251细胞增殖有显著抑制作用,在10μmol·L-1～100μmol·L-1浓度范围内呈剂量和作用时间依赖性,作用48h和72h的IC50分别为55.33μmol·L-1,52.7μmol·L-1。60μmol·L-1 BVAN08作用12h后,U-251细胞产生明显的G2／M期阻滞(63.3%),并开始同时出现细胞凋亡和自吞噬死亡。BVAN08对U-251细胞有明显的放射增敏作用,而且在照射前12h给药的增敏效果最好,20μmol·L-1浓度对2 Gy照射杀伤U-251细胞的增强比为3.14。Western blot检测表明BVAN08能显著抑制DNA-PKcs的表达。四、结论6-溴异香兰素具有明显抑制人脑胶质瘤细胞增殖和辐射增敏作用,能诱发肿瘤细胞凋亡和自吞噬死亡。其能从两个方面发挥抗癌活性,即对癌细胞产生的直接杀伤和抗增殖效应以及对放射治疗增敏效应,并且其辐射增敏效应可能与G2／M期阻滞和DNA双链断裂修复关键蛋白DNA-PKcs表达的抑制有关。结果显示了BVAN08是一种很有开发前景的抗肿瘤药物。更多还原

【Abstract】 ObjectiveTo investigate effects of vanillin derivative bromoisovanillin (6-bromine-5-hydroxyl-4-methoxy-benzaldehyde, BVAN08) on the growth inhibition, the radiosensitization of human glioma cell line U-251, and the relative mechanism to provide further convincing evidences and experimental data for exploring BVAN08 as a new anticancer drug.MethodsMTT was performed to analyze the cytotoxicity and proliferation inhibitive effect of BVAN08; colony-forming ability assay was used to determine the survival curve of U-251 cells exposed to different doses of 60Coγirradiation, the radiosensitization of U-251 cells by BVAN08-which treated cells before the irradiation or after irradiation. Flow cytometry was used to detect the cell cycle changes and apoptosis induction; Cellular morphology changes were observed by light microscope; and the autophagic cell death was observed by TEM (transmission electron microscope); Expression changes of DNA-PKcs protein were detected by Western blot.ResultsThe MTT analysis data indicated an dose-dependently and time-dependently inhibitive effect of BVAN08 on the proliferation of glioma U-251 cells at the concentrations from 10μmol·L-1 to 100μmol·L-1; The IC50 of BVAN08 treatment for 48h and 72h were 55.33μmol·L-1 and 52.70μmol·L-1 respectively; An obvious G2/M arrest (63.3%) was observed in U-251 cells after 12h treatment with 60μmol·L-1 BVAN08 and apoptosis and autophagic cell death were also triggered at the same time. BVAN08 shows obvious radiosensitization on U-251 cells, and the most effective sensitizing effect was obtained when cells were pretreated with BVAN08 12h before irradiation. The enhanced ratio of radiosensitization (cell killing) by 20μmol·L-1 BVAN08 on 2 Gy irradiation is 3.14. Western blotting analysis showed the expression of DNA-PKcs was obviously inhibited by BVAN08.ConclusionsBVAN08 inhibits the proliferation of human glioma cells via inducing apoptosis and autophygic cell death, and exhibiting radiosensitizing effect. The anticancer activity of BVAN08 could be fulfilled through two aspects, killing the cancer cells directly and sensitizing cancer cells to radiotherapy. The radiosensitization of BVAN08 may be related with G2/M arrest and the inhibition of DNA-PKcs expression which is a critical component involved in the non-homologous end joining pathway of DNA double-strand breaks repair.更多还原