唑来膦酸抑制结直肠癌细胞增殖的分子机制研究

发布时间：2018-12-14 20:16

【摘要】：目的:探讨唑来膦酸对HCT116细胞的生物学作用及其分子机制。方法:1)采用CCK-8方法检测不同浓度唑来膦酸对HCT116细胞增殖的作用,不同浓度唑来膦酸孵育HCT116细胞72h后,测定细胞存活率,并计算IC50值;2)采用克隆形成实验观察唑来膦酸对HCT116细胞克隆形成能力的影响;3)采用流式细胞分析术分析25μmol/L和50μmol/L的唑来膦酸作用HCT116细胞48h后细胞的凋亡比例;4)25μmol/L和50μmol/L唑来膦酸作用HCT116细胞24h、48h和72h后,用线粒体膜电位特异性染料JC-1避光孵育细胞15min,采用荧光显微镜和流式细胞术分析其红色荧光强度的变化;5)Western blot方法分析唑来膦酸作用HCT116细胞72h后,细胞线粒体内和胞浆内cytochrome C含量的改变以及胞浆内caspase-3的活化情况;6)建立HCT116细胞裸鼠移植瘤模型,并观测给药唑来膦酸后肿瘤生长、瘤块重量及肿瘤组织形态学的变化。结果:1)唑来膦酸可抑制HCT116细胞的增殖,并具有剂量依赖性,IC50=26.79μmol/L;2)IC50浓度的唑来膦酸可显著抑制HCT116细胞克隆的形成;3)唑来膦酸25μmol/L和50μmol/L作用HCT116细胞48h后,HCT116细胞发生凋亡,凋亡比例分别为11.6%±0.5%、49.2%±3.4%,p0.05;4)25μmol/L和50μmol/L唑来膦酸作用HCT116细胞24h、48h和72h后,荧光显微镜观察结果显示JC-1被激发出的红色荧光强度显著降低,流式细胞术分析结果显示被激发出JC-1红色荧光的细胞比例显著减少:25μmol/L唑来膦酸作用HCT116细胞24h、48h和72h后,JC-1红色荧光细胞的比例分别为96.49%±2.1%、86.13%±3.2%、74.23%±5.3%,p0.05;50μmol/L唑来膦酸作用HCT116细胞24h、48h和72h后,JC-1红色荧光细胞的比例分别为55.21%±5.9%、66.65%±2.3%、38.71%±4.3%,p0.05;5)Western blot结果显示25μmol/L和50μmol/L唑来膦酸作用HCT116细胞72h后,线粒体中cytochrome C减少,胞浆中cytochrome C增多,同时,活化的caspase 3增多。说明cytochrome C从线粒体内释放至胞浆,并最终激活了Caspase-3;6)体内实验结果显示2mg/kg唑来膦酸治疗可显著抑制小鼠肿瘤生长,末次测量肿瘤体积对照组为2289.4±363.8mm3,唑来膦酸组为926.42±238.3mm3;试验结束后剥离肿瘤组织,对照组、2mg/kg唑来膦酸治疗组的平均瘤重分别为2.53±0.33g、1.65±0.31g,唑来膦酸组肿瘤体积显著小于对照组,抑瘤率为:34.8%;移植瘤的组织形态学检查显示唑来膦酸诱导了肿瘤细胞凋亡。结论:唑来膦酸能抑制HCT116细胞增殖和克隆形成,其机制是通过使线粒体跨膜电位下降,通透性增强,导致cytochrome C由线粒体释放至细胞浆,最终激活了caspase-3,导致细胞凋亡。动物实验证明唑来膦酸可抑制体内肿瘤生长,诱导肿瘤组织发生凋亡。该研究为ZOL作为结直肠癌治疗药物的开发与临床应用提供了理论依据。
[Abstract]:Aim: to investigate the biological effect of zoledronic acid on HCT116 cells and its molecular mechanism. Methods: 1) the effects of zoledronic acid at different concentrations on the proliferation of HCT116 cells were detected by CCK-8 method. After incubated with zoledronic acid for 72 hours, the survival rate of HCT116 cells was measured and the IC50 value was calculated. 2) the effect of zoledronic acid on the clonal formation of HCT116 cells was observed by clone formation assay, 3) the apoptotic ratio of HCT116 cells treated with 25 渭 mol/L and 50 渭 mol/L zoledronic acid for 48 h was analyzed by flow cytometry. 4) after treated with 25 渭 mol/L and 50 渭 mol/L zoledronic acid for 24 h and 72 h, the cells were incubated with mitochondrial membrane potential specific dye JC-1 for 15 min. The changes of red fluorescence intensity were analyzed by fluorescence microscopy and flow cytometry. 5) Western blot method was used to analyze the changes of cytochrome C content in mitochondria and cytoplasm and the activation of caspase-3 in cytoplasm of HCT116 cells treated with zoledronic acid for 72 hours. 6) the model of transplanted tumor of HCT116 cells in nude mice was established, and the tumor growth, tumor mass weight and tumor histomorphology were observed after administration of azoledronic acid. Results: 1) Zoledronic acid inhibited the proliferation of HCT116 cells in a dose-dependent manner, and zoledronic acid at the concentration of IC50=26.79 渭 mol/L;2 significantly inhibited the clone formation of HCT116 cells. 3) after treated with 25 渭 mol/L and 50 渭 mol/L of zoledronic acid for 48 h, the apoptosis of HCT116 cells was 11.6% 卤0.5% and 49.2% 卤3.4%, respectively. 4) after treatment of HCT116 cells with 25 渭 mol/L and 50 渭 mol/L zoledronic acid for 48 h and 72 h, the results of fluorescence microscopy showed that the red fluorescence intensity induced by JC-1 was significantly decreased. The results of flow cytometry showed that the percentage of cells stimulated with JC-1 red fluorescence was significantly decreased: the percentage of JC-1 red fluorescent cells was 96.49% 卤2.1 after treatment with 25 渭 mol/L zoledronic acid for 24 h and 72 h, respectively. 86.13% 卤3.2% and 74.23% 卤5.3%. After treatment with 50 渭 mol/L zoledronic acid for 24 h or 72 h, the percentage of JC-1 red fluorescent cells was 55.21% 卤5.9% 卤6.65% 卤2.3% and 38.71% 卤4.3% respectively. 5) Western blot results showed that after treated with 25 渭 mol/L and 50 渭 mol/L zoledronic acid for 72 h, cytochrome C in mitochondria decreased, cytochrome C in cytoplasm increased and activated caspase 3 increased. The results showed that cytochrome C was released from mitochondria to cytoplasm and Caspase-3; was activated. 6) the results of in vivo experiments showed that 2mg/kg zoledronic acid could significantly inhibit tumor growth in mice. The tumor volume in the last tumor control group was 2289.4 卤363.8mm ~ (-3) mm ~ (-3), and that in the zoledronic acid group was 926.42 卤238.3 mm ~ (-3). The mean tumor weight of the control group and 2mg/kg zoledronic acid treatment group was 2.53 卤0.33 g / L 1.65 卤0.31 g respectively, and the tumor volume of zoledronic acid group was significantly smaller than that of the control group (34.8%). Histological examination of transplanted tumor showed that zoledronic acid induced apoptosis of tumor cells. Conclusion: zoledronic acid can inhibit the proliferation and clone formation of HCT116 cells through the decrease of mitochondrial transmembrane potential and the enhancement of permeability, resulting in the release of cytochrome C from mitochondria to the cytoplasm and the activation of caspase-3, to induce apoptosis. Animal experiments have shown that zoledronic acid can inhibit tumor growth and induce apoptosis in tumor tissues. This study provides a theoretical basis for the development and clinical application of ZOL as a therapeutic drug for colorectal cancer.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.34

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