塞来昔布对胶质瘤U251细胞增殖、凋亡和Survivin表达的影响

Effect of celecoxib on proliferation,apoptosis,and Survivin expression in human glioma cell line U251

背景与目的：选择性COX-2抑制剂塞来昔布是近年来开发的新型非甾体类抗炎药。大量实验证明塞来昔布具有明显的抑制肿瘤细胞增殖和诱导凋亡的作用,这在结直肠肿瘤和家族性腺瘤性息肉病中已经得到了证实。本研究观察塞来昔布在体外对人胶质瘤细胞株U251的抑制增殖和诱导凋亡的作用,并探讨该作用与Survivin表达之间的关系。方法：用不同浓度塞来昔布溶液处理对数生长期的U251细胞,在倒置显微镜下动态观察瘤细胞的生长情况。用MTT比色法检测不同时间点各药物浓度组细胞增殖情况。不同浓度塞来昔布处理U251细胞48h后,用流式细胞技术检测各组细胞的凋亡率,免疫细胞化学法及Westernblot法检测各组细胞中Survivin蛋白的表达情况,逆转录聚合酶链反应（RT-PCR）半定量法检测各组细胞中survivinmRNA的表达水平。结果：塞来昔布处理后细胞出现明显凋亡样形态学改变。10~100μmol/L塞来昔布对U251细胞增殖均有抑制作用,该作用随着药物浓度的增加和作用时间的延长而增强。塞来昔布处理U251细胞48h后,流式细胞检测结果显示出明显的细胞凋亡,凋亡率随着塞来昔布浓度的增高而升高;免疫细胞化学结果显示,对照组细胞中Survivin蛋白高表达,实验组中随着塞来昔布浓度的增加阳性细胞数目逐渐减少、染色逐渐减淡、蛋白表达的灰度值亦随之上升;Westernblot结果显示随着药物浓度的增高,Survivin蛋白表达量逐渐降低;半定量RT-PCR检测结果显示,塞来昔布作用后U251细胞中survivinmRNA表达明显降低,各实验组与对照组之间的差异均有统计学意义（P〈0.05）。结论：塞来昔布可明显抑制胶质瘤U251细胞增殖并促使瘤细胞凋亡,这些作用呈时间和剂量依赖性。塞来昔布抗肿瘤的作用机制可能与下调survivin的表达有关。

Background and Objective： Celecoxib,one of the new generation of non-steroidal anti-inflammatory drugs （NSAIDs）,has a specific inhibitory effect on COX-2. Studies have shown that celecoxib can inhibit the proliferation of tumor cells and induce cell apoptosis,which has been confirmed in colorectal tumors and familial adenomatous polyposis. This study explored the effect of celecoxib on the proliferation and apoptosis of human glioma cell line U251 and elucidated the correlation between the effect of celecoxib and the expression of survivin. Methods： U251 cells were treated with different concentrations of celecoxib. Cell morphologic changes were observed by optical microscopy. MTT assay was used to detect the absorbance value and to calculate inhibition and survival rates. The rates of apoptosis of U251 cells after 48 h of treatment with celecoxib were assessed by flow cytometry. The expression of survivin was analyzed by immunocytochemistry （ICC） and Western blot analysis. The expression of survivin mRNA was determined by reverse transcription-polymerase chain reaction （RT-PCR）. Results： Significant morphologic changes were shown in U251 cells after treatment with celecoxib. The MTT assay results revealed that celecoxib inhibited the proliferation of U251 cells and the inhibitory rates significantly increased in a dose- and time-dependent manner. After 48 h of treatment with celecoxib,the apoptotic cells could be obviously observed,and the apoptosis rate significantly increased with increases in concentrations of celecoxib. The expression of survivin was observed in the control group,however,the expression of survivin was significantly down-regulated as the concentration of celecoxib increased. The level of survivin mRNA expression in U251 cells was significantly down-regulated after treatment with different concentrations of celecoxib （ P〈0.05）. Conclusion： The inhibition of proliferation and apoptosis in U251 cells could be induced by celecoxib in a dose- and time-dependent manner,and its mechanism might be the downregulation of the expression of survivin.