摘要
目的:观察华蟾酥毒基对人骨肉瘤细胞系U2OS增殖和凋亡的影响,从而探讨华蟾酥毒基诱导骨肉瘤凋亡的可能机制。方法:体外培养人骨肉瘤细胞系U2OS,CCK-8法检测不同浓度华蟾酥毒基对骨肉瘤细胞系U2OS增殖的抑制作用,Annexin V-FITC/PI染色,流式细胞术检测细胞凋亡,Hoechst 33258染色,显微镜下观察细胞凋亡的形态学变化,应用逆转录荧光定量PCR法检测华蟾酥毒基对凋亡通路相关mRNA表达的影响。结果:CCK-8检测显示华蟾酥毒基可抑制人骨肉瘤U2OS细胞增殖,并具有时间和剂量依赖性,在华蟾酥毒基浓度为20 nmol·L^(-1)时,对细胞增殖的抑制作用明显增强;流式细胞术检测显示华蟾酥毒基可明显诱导骨肉瘤细胞凋亡;Hoechst 33258染色可见凋亡小体;逆转录荧光定量PCR分析显示经华蟾酥毒基处理后的骨肉瘤细胞系U2OS表达Caspase-3,8,9较用药前明显增高。结论:华蟾酥毒基对骨肉瘤细胞系U2OS的增殖有显著抑制作用,并可诱导其发生凋亡,其作用机制可能与激活Caspase依赖的凋亡途径有关。
Objective： To investigate the effect of cinobufagin on the growth inhibition, apoptosis and the expression of Caspases mRNA in human osteosarcoma cell line U2OS. Methods： Osteosarcoma cell line U2OS was cultured in vitro. Cell proliferation was detected by Cell Counting Kit-8 （ CCK-8 ）. The cells stained by Annexin V and PI were observed by flow cytometry （FCM） for apoptosis. Hoechst 33258 was used to detect the morphological change of apoptotic cells. The mRNA ex- pression of Caspases in U2OS was observed by RT-PCR. Results： The proliferation of human osteosarcoma cell line U2OS could be inhibited and apoptosis could be induced by cinobufagin. The inhibition rate and apoptosis rate were different with the different concentrations. The inhibition rate and apoptosis rate of U2OS cells rose significantly when the concentration of cinob- ufagin was over 20 nmol · L^-1. Apoptotic body could be observed by Hoechst 33258 dying. The Caspases mRNA expression of human osteosarcoma cell line U20S increased obviously by using cinobufagin. Conclusion： Cinobufagin could significantly inhibit the proliferation and induce apoptosis of human osteosarcoma cell line U2OS. Its mechanism of action may be related to activation of Caspase dependent apoptosis pathway.
出处
《中国现代中药》
CAS
2017年第9期1249-1253,共5页
Modern Chinese Medicine
基金
国家自然科学基金(81341078)
