尼美舒利诱导人胶质瘤U87细胞凋亡的机制

发布时间：2018-10-15 16:44

【摘要】：目的：本实验拟通过MTT法、吖啶橙（AO）/溴乙啶(EB）荧光染色法、流式细胞术、Western印迹等方法，观察尼美舒利在体外对胶质瘤U87细胞增殖和凋亡的影响，并对其作用机制进行初步探讨。方法：取胶质瘤U87细胞培养18小时，根据尼美舒利作用的不同浓度分组：0.05、0.1、0.2、0.4、0.8mmol/L，不同的作用时间分组：12、24、48h；然后采用四甲基耦氮唑蓝（MTT）法计算胶质瘤U87细胞抑制率；吖啶橙（AO）/溴乙啶（EB）荧光染色法和AnnexinV-FITC/PI双染法流式细胞术检测U87细胞凋亡率的变化；最后使用Westem印迹法观察U87细胞中Cox-2、Bcl-2和Bax蛋白表达的变化。结果：（1）MTT法分析，发现用含不同浓度的尼美舒利溶液分别培养U87细胞12、24、48小时后，细胞生长被明显抑制，且与药物作用时间-浓度正相关。（2）吖啶橙（AO）/溴乙啶(EB）荧光染色和透射式荧光显微镜观察显示尼美舒利使胶质瘤U87细胞发生显著的凋亡形态改变；进一步应用AnnexinV-FITC/PI双染法流式细胞仪分析结果表明，尼美舒利主要使U87细胞发生在晚期凋亡，且随尼美舒利浓度的增加细胞凋亡率明显上升。（3）Western印迹凝胶电泳法发现用0.2、0.4、0.8mmol/L的尼美舒利溶液作用于U87细胞24小时后，尼美舒利不仅能剂量性地降低U87细胞中Bcl-2、Cox-2蛋白的表达，同时也可剂量依赖性地升高Bax蛋白的表达。结论：（1）尼美舒利在体外能有效地抑制胶质瘤U87细胞的生长，且细胞抑制率呈现时间-剂量依赖性；（2）尼美舒利能促进胶质瘤U87细胞凋亡，，多发生在晚期，且细胞凋亡率呈剂量依赖性；（3）尼美舒利在体外能显著抑制胶质瘤U87细胞的增殖并诱导其凋亡，其作用机制可能是通过抑制Cox-2蛋白和下调Bcl-2蛋白的表达，升高Bax蛋白的表达来实现其抗肿瘤作用。
[Abstract]:Aim: to observe the effect of nimesulide on the proliferation and apoptosis of U87 cells in vitro by MTT, acridine orange (AO) / ethidium (EB) fluorescence staining, flow cytometry and Western blot, and to explore its mechanism. Methods: U87 glioma cells were cultured for 18 hours. According to the different concentrations of nimesulide, the cells were divided into 0. 05 mmol / L, 0. 2, 0. 4 and 0. 8 mmol / L, respectively. The inhibitory rate of U87 cells was calculated by tetramethyl azolium blue (MTT) assay. The apoptosis rate of U87 cells was detected by flow cytometry with acridine orange (AO) / ethidium bromide (EB) fluorescent staining and AnnexinV-FITC/PI double staining, and the expression of Cox-2,Bcl-2 and Bax in U87 cells was observed by Westem blot. Results: (1) MTT assay showed that the growth of U87 cells cultured with different concentrations of nimesulide for 48 hours was significantly inhibited. (2) fluorescence staining of acridine orange (AO) / bromoethidime (EB) and transmission fluorescence microscopy showed that nimesulide induced apoptosis in U87 cells. The results of AnnexinV-FITC/PI double staining flow cytometry showed that nimesulide mainly caused U87 cell apoptosis in the late stage. The apoptotic rate of U87 cells increased with the increase of nimesulide concentration. (3) Western blot gel electrophoresis showed that nimesulide not only decreased the expression of Bcl-2,Cox-2 protein in U87 cells, but also decreased the expression of Bcl-2,Cox-2 protein in U87 cells after 24 hours treated with 0.2nil 0.4nmol / L nimesulide solution. At the same time, the expression of Bax protein was increased in a dose-dependent manner. Conclusion: (1) nimesulide can effectively inhibit the growth of U87 glioma cells in vitro, and the inhibition rate is time-dose dependent, (2) nimesulide can promote the apoptosis of U87 glioma cells, most of them occur in the late stage. (3) nimesulide could significantly inhibit the proliferation and induce the apoptosis of U87 glioma cells in vitro by inhibiting the expression of Cox-2 protein and down-regulating the expression of Bcl-2 protein. Increase the expression of Bax protein to achieve its anti-tumor effect.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

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