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目的研究槲皮素(quercetin)诱导HeLa细胞(人宫颈癌细胞)凋亡的作用,以及caspase-3、caspase-8活化对凋亡的影响。方法采用噻唑蓝(MTT)法观察槲皮素对HeLa细胞(人宫颈癌细胞)和L-02细胞(正常人肝细胞)的生长抑制作用;DA-PI染色后荧光显微镜观察细胞的形态学变化;通过DNA琼脂糖凝胶电泳进行DNA片段化分析,FITC-Annexin V/PI荧光标记流式细胞术检测细胞凋亡情况;比色法测定槲皮素诱导HeLa细胞凋亡过程中caspase-3、caspase-8活性变化。结果 MTT结果显示,槲皮素可选择性抑制HeLa细胞的生长,并呈剂量、时间依赖性,而正常人肝细胞L-02不出现生长抑制(P>0.05)。160.0μmol.L-1槲皮素处理HeLa细胞72 h,DAPI染色也可见核浓缩及边缘现象,DNA电泳出现特征性的凋亡条带。160.0μmol.L-1槲皮素处理HeLa细胞24、48和72 h的凋亡率分别为(9.4±2.1)%、(30.1±6.5)%和(59.8±9.5)%,caspase-3活性在48 h达最高(1.68±0.07)U.μg-1,而caspase-8活性则在24 h达最高(1.83±0.06)U.μg-1,两者与对照组相比均具有显著性差异(P<0.05)。结论槲皮素可特异性地诱导人HeLa细胞凋亡,其诱导凋亡的机制可能与caspase-3、caspase-8活化有关。
Objective To study the effect of quercetin on the apoptosis of HeLa cells (human cervical cancer cells) and the effect of caspase-3 and caspase-8 activation on apoptosis. Methods The inhibitory effect of quercetin on the growth of HeLa cells (human cervical cancer cells) and L-02 cells (normal human hepatocytes) was observed by MTT assay. The morphological changes of cells were observed under light microscope after DA-PI staining ; DNA fragmentation was analyzed by DNA agarose gel electrophoresis; FITC-Annexin V / PI fluorescence-labeled flow cytometry was used to detect the apoptosis; and the content of caspase-3 in quercetin-induced HeLa cell apoptosis was measured by colorimetry. Changes in caspase-8 activity. Results MTT results showed that quercetin could selectively inhibit the growth of HeLa cells in a dose-and time-dependent manner, while no inhibition of growth was observed in normal human hepatocytes L-02 (P> 0.05). HeLa cells were treated with 160.0 μmol·L-1 quercetin for 72 h. DAPI staining also showed nuclear condensation and marginal phenomena. A characteristic apoptotic band appeared in DNA electrophoresis. The apoptotic rates of HeLa cells treated with 160.0 μmol·L-1 quercetin for 24, 48 and 72 h were (9.4 ± 2.1)%, (30.1 ± 6.5)% and (59.8 ± 9.5)%, respectively. The caspase- (1.68 ± 0.07) U.μg-1 at 48 h and caspase-8 activity at 24 h reached the highest (1.83 ± 0.06) U.μg-1, both of which had significant difference compared with the control group P <0.05). Conclusion Quercetin can specifically induce apoptosis in human HeLa cells. The mechanism of apoptosis may be related to the activation of caspase-3 and caspase-8.