摘要
目的研究芸香诱导白血病K562细胞凋亡过程中Caspase-3基因的表达。方法体外培养白血病K562细胞,生长曲线观察芸香对K562细胞的细胞抑制作用,MTT法观察芸香的细胞毒作用,测定IC50,Hoechst33342/PI双荧光染色后,荧光显微镜下观察细胞核形态,流式细胞仪检测细胞周期,RT-PCR观察Caspase-3的表达,CaspACETM分析系统检测Caspase-3酶活性。结果芸香浓度为2×10-5mol/L、4×10-5mol/L、8×10-5mol/L时,细胞生长受到显著抑制;不同浓度quercetin处理K562细胞48h后,IC50为4×10-5mol/L;Hoechst33342/PI双荧光染色可观察到明显核固缩、凝集等细胞凋亡表现;流式细胞仪检测芸香处理后细胞发现G2期细胞显著增多(64.7%),DNA合成受抑,凋亡率增加;以Caspase-3基因和内参照β-actin序列设计引物,PCR产物经过琼脂糖凝胶电泳,紫外透射仪下明显观察到约300bp条带;CaspACETM分析系统检测Caspase-3酶活性明显升高(P<0.05)。结论芸香诱导白血病细胞Caspase-3基因表达,细胞发生凋亡,caspase-3途径诱导凋亡是芸香作用机制之一。
Objective To study the mechanism of quercetin induced the expression of Caspase-3 in leukemia cell Methods K562 cells was cultured in vitro and then studied by MTT to measure IC50, so as to observe the cytotoxicity of quercetin; The morphology changes of the nucleus were observed by Hoechst33342/PI staining, flow cytometry（FCM） showed the cell cycle, RT-PCR was used to amplify cDNA copies of RNA, amplification was observed by UV light, so as to measure the expression of Caspase-3 in the process of quercetin induced K562 apoptosis. Caspase^TM analysis system was used to measure Caspase-3. Results When the concentrations of quercetin were 2×10^-5 mol/L, 4×10^-5 mol/ L, 8×10^-5 mol/L, the cell growth was inhibited remarkably in the way of dosage dependence. After the K562 cells were treated by different concentrations of quercetin for 48 hours, the IC50 was 4×10^-5 mol/L by MTT experiments, distinct morphologic changes of cell apoptosis such as karyopyknosis and conglomeration were observed by Hoechst3334/PI staining. FCM showed the number of G2 phase cell increased （64.7 %）. The synthesis of DNA was inhibited, and the rate of apoptotic cells was elevated. PCR products showed the expression of Caspase-3 after 24 hours. CaspaceTM analysis system showed higher Caspase-3 （P 〈 0.05）. Conclusion Quercetin exerts its anti-cancer effect by inducing the leukemia cell apoptosis, and Up-regulated expression of Caspase-3 may be one of the mechanisms.
出处
《中国中西医结合外科杂志》
CAS
2006年第6期575-579,共5页
Chinese Journal of Surgery of Integrated Traditional and Western Medicine
