塞来昔布诱导喉癌Hep-2细胞凋亡的实验研究

Apoptosis of Hep-2 cells induced by celecoxib

目的探讨环氧化酶-2特异抑制剂——塞来昔布对喉癌Hep-2细胞增殖抑制和凋亡诱导作用及可能机制。方法以不同浓度的塞来昔布处理喉癌Hep-2细胞,以四甲基偶氮唑盐(MTT)法检测对细胞增殖的影响,Hoechst33342染色荧光显微镜下观察细胞核的形态改变,以Annexin V/PI双染法检测细胞的凋亡率,Western blot检测Bcl-2、Bax及COX-2的表达,Western blot分析Caspase-3裂解激活。结果 MTT结果表明塞来昔布抑制Hep-2细胞的增殖呈时间和浓度依赖;Hoechst33342荧光染色见随塞来昔布处理时间延长,细胞核逐渐出现凝集、碎裂等凋亡表现;流式细胞术检测细胞凋亡率见塞来昔布处理细胞24 h,70、100μmol/L组凋亡率分别为(28.9±2.74)%、(32.7±3.96)%;Western blot分析蛋白表达,见随药物处理浓度增加,COX-2、Bcl-2表达降低,Bax表达增加,随药物处理时间延长Caspase-3出现裂解片段。结论塞来昔布能够有效地抑制Hep-2细胞增殖和诱导Hep-2细胞凋亡,其诱导凋亡作用机制可能与Bax/Bcl-2比值升高,Caspase-3蛋白裂解激活有关。

Objective To investigate the mechanism of celecoxib, a COX-2 inhibitor, in inducing cell apoptosis in hu man laryngocarcinoma Hep-2 cells. Methods MTT was used to observe the proliferation of Hep-2 cells treated with various doses of celecoxib. Cell apoptosis was determined by flow cytometry and morphologic observation under fluo rescent microscopy with Hoechst33258 staining. Protein expressions of Bax, Bcl-2, COX-2 were examined by western blot. Caspase-3 activation was examined by western blot. Results M＇Fr results showed that celecoxib could inhibit the proliferation of Hep-2 cells in a time- and concentrationdependent manner. Hoechst33342 fluorescent staining showed the nucleus gradually appeared agglutination and fragmentation after the treatment of celecoxib. Apoptosis rate in the groups treated with 70 and 100 μ mol/L celecoxib were （ 28.9 ± 2.74 ） % and （ 32.7 ± 3.96 ） % correspondingly. Wwestern blot showed that celecoxib could down-regulate the levels of expressions of COX-2 and Bcl-2 protein, up-reg- ulate Bax protein＇ s expression, and activate caspase-3. Conclusion Celecoxib can effectively inhibit the proliferation of Hep-2 cells and induce the apoptosis of Hep-2 cells. The induction of apoptosis may be related to the increased Bax/Bcl-2 ratio and activation of caspase-3.