中华骨科杂志
中華骨科雜誌
중화골과잡지
CHINESE JOURNAL OF ORTHOPAEDICS
2014年
4期
472-477
,共6页
尹军强%谢显彪%温丽丽%黄纲%王博%王晋%沈靖南
尹軍彊%謝顯彪%溫麗麗%黃綱%王博%王晉%瀋靖南
윤군강%사현표%온려려%황강%왕박%왕진%침정남
华蟾蜍毒素%骨肉瘤%细胞凋亡
華蟾蜍毒素%骨肉瘤%細胞凋亡
화섬서독소%골육류%세포조망
Cinobufagin%Osteosarcoma%Apoptosis
目的 研究华蟾酥毒基(cinobufagin)对多种骨肉瘤细胞系的增殖抑制和诱导凋亡作用,并探讨其相关机制.方法 不同浓度的华蟾素毒基分别处理骨肉瘤细胞系U2OS、MG63和SaO2后,四甲基偶氮唑蓝(MTT)法观察不同骨肉瘤细胞系的增殖活性,流式细胞仪检测细胞周期的变化,细胞核荧光染色及Annexin V/PI染色检测细胞凋亡,Western blot检测IAPs和Bcl-2家族凋亡相关蛋白凋亡相关蛋白Bax、cleaved-PARP、xIAP、cIAP-1、survivin及p65的表达.结果 MTT检测显示,华蟾酥毒基可明显抑制骨肉瘤细胞U2OS、MG63和SaO2的增殖,其抑制细胞增殖的作用呈剂量和时间依赖性,其对U2OS、MG63和SaO2细胞的48 h IC50值分别为(104.83±16.96) nmol/L、(47.07±7.5) nmol/L和(136.72±10.08)nmol/L.100 nmol/L华蟾酥毒基处理骨肉瘤细胞12 h后,流式细胞仪检测可见骨肉瘤U2OS、MG63和SaO2的GdG1期细胞比例下降,GJM期细胞比例增加,表明华蟾素毒基主要通过将细胞阻滞在GJM期,以抑制骨肉瘤细胞的增殖.进一步Hoechst 33258细胞核染色发现,华蟾素毒基可诱导骨肉瘤细胞产生典型凋亡小体,Annexin V/PI检测100 nmol/L华蟾酥毒基作用48 h后,U2OS、MG63和SaO2细胞凋亡率分别为33.6％±6.4％、36.4％±7.8％及29.3％±5.1％.表明华蟾酥毒基的抗骨肉瘤效果是通过诱导骨肉瘤细胞凋亡来实现.在探讨其诱导骨肉瘤细胞凋亡的相关机制中,通过Western blot检测发现其诱导凋亡的作用机制是通过上调IAPs和Bcl-2家族凋亡相关蛋白Bax、cleaved-PARP,下调xIAP、cIAP-1、survivin及p65蛋白来实现.结论 华蟾酥毒基可明显抑制骨肉瘤细胞株U2OS、MG63和SaO2细胞增殖,阻滞细胞在G2/M期,并诱导其凋亡,其诱导凋亡的作用机制为调节IAPs和Bcl-2凋亡相关家族蛋白的活性.
目的 研究華蟾酥毒基(cinobufagin)對多種骨肉瘤細胞繫的增殖抑製和誘導凋亡作用,併探討其相關機製.方法 不同濃度的華蟾素毒基分彆處理骨肉瘤細胞繫U2OS、MG63和SaO2後,四甲基偶氮唑藍(MTT)法觀察不同骨肉瘤細胞繫的增殖活性,流式細胞儀檢測細胞週期的變化,細胞覈熒光染色及Annexin V/PI染色檢測細胞凋亡,Western blot檢測IAPs和Bcl-2傢族凋亡相關蛋白凋亡相關蛋白Bax、cleaved-PARP、xIAP、cIAP-1、survivin及p65的錶達.結果 MTT檢測顯示,華蟾酥毒基可明顯抑製骨肉瘤細胞U2OS、MG63和SaO2的增殖,其抑製細胞增殖的作用呈劑量和時間依賴性,其對U2OS、MG63和SaO2細胞的48 h IC50值分彆為(104.83±16.96) nmol/L、(47.07±7.5) nmol/L和(136.72±10.08)nmol/L.100 nmol/L華蟾酥毒基處理骨肉瘤細胞12 h後,流式細胞儀檢測可見骨肉瘤U2OS、MG63和SaO2的GdG1期細胞比例下降,GJM期細胞比例增加,錶明華蟾素毒基主要通過將細胞阻滯在GJM期,以抑製骨肉瘤細胞的增殖.進一步Hoechst 33258細胞覈染色髮現,華蟾素毒基可誘導骨肉瘤細胞產生典型凋亡小體,Annexin V/PI檢測100 nmol/L華蟾酥毒基作用48 h後,U2OS、MG63和SaO2細胞凋亡率分彆為33.6％±6.4％、36.4％±7.8％及29.3％±5.1％.錶明華蟾酥毒基的抗骨肉瘤效果是通過誘導骨肉瘤細胞凋亡來實現.在探討其誘導骨肉瘤細胞凋亡的相關機製中,通過Western blot檢測髮現其誘導凋亡的作用機製是通過上調IAPs和Bcl-2傢族凋亡相關蛋白Bax、cleaved-PARP,下調xIAP、cIAP-1、survivin及p65蛋白來實現.結論 華蟾酥毒基可明顯抑製骨肉瘤細胞株U2OS、MG63和SaO2細胞增殖,阻滯細胞在G2/M期,併誘導其凋亡,其誘導凋亡的作用機製為調節IAPs和Bcl-2凋亡相關傢族蛋白的活性.
목적 연구화섬소독기(cinobufagin)대다충골육류세포계적증식억제화유도조망작용,병탐토기상관궤제.방법 불동농도적화섬소독기분별처리골육류세포계U2OS、MG63화SaO2후,사갑기우담서람(MTT)법관찰불동골육류세포계적증식활성,류식세포의검측세포주기적변화,세포핵형광염색급Annexin V/PI염색검측세포조망,Western blot검측IAPs화Bcl-2가족조망상관단백조망상관단백Bax、cleaved-PARP、xIAP、cIAP-1、survivin급p65적표체.결과 MTT검측현시,화섬소독기가명현억제골육류세포U2OS、MG63화SaO2적증식,기억제세포증식적작용정제량화시간의뢰성,기대U2OS、MG63화SaO2세포적48 h IC50치분별위(104.83±16.96) nmol/L、(47.07±7.5) nmol/L화(136.72±10.08)nmol/L.100 nmol/L화섬소독기처리골육류세포12 h후,류식세포의검측가견골육류U2OS、MG63화SaO2적GdG1기세포비례하강,GJM기세포비례증가,표명화섬소독기주요통과장세포조체재GJM기,이억제골육류세포적증식.진일보Hoechst 33258세포핵염색발현,화섬소독기가유도골육류세포산생전형조망소체,Annexin V/PI검측100 nmol/L화섬소독기작용48 h후,U2OS、MG63화SaO2세포조망솔분별위33.6％±6.4％、36.4％±7.8％급29.3％±5.1％.표명화섬소독기적항골육류효과시통과유도골육류세포조망래실현.재탐토기유도골육류세포조망적상관궤제중,통과Western blot검측발현기유도조망적작용궤제시통과상조IAPs화Bcl-2가족조망상관단백Bax、cleaved-PARP,하조xIAP、cIAP-1、survivin급p65단백래실현.결론 화섬소독기가명현억제골육류세포주U2OS、MG63화SaO2세포증식,조체세포재G2/M기,병유도기조망,기유도조망적작용궤제위조절IAPs화Bcl-2조망상관가족단백적활성.
Objective To study the growth inhibition,apoptosis induction effects of cinobufagin(CB)on human osteosarcoma(OS) cell line U2OS,MG63 and SaO2 in vitro and the underlying mechanism of action of cinobufagin in OS cells.Methods Cell viability was assessed by MTT assay.Cell-cycle status,apoptosis-inducing effects were evaluated by flow cytometry,fluorescent staining and DNA fragmentation assays.Inhibitors of apoptosis proteins (IAPs) and Bcl-2 family proteins including Bax,cleaved-PARP,xIAP,cIAP-1,survivin and p65 were tested by Western blot.Results MTT assay showed that CB could inhibited the growth of U2OS,MG63 and SaO2 cells in a dose-and time-dependent manner.The 48 h IC50 of CB on U2OS,MG63 and SaO2 cells were (104.83± 16.96) nmol/L,(47.07±7.5) nmol/L,and (136.72±10.08) nmol/L respectively.The induction of G2/M cell-cycle arrest was seen in the cells treated with CB.After cells were cultured for 12 h in the presence of 100 nmol/L CB,the percentages of cells in the G0/G1 phase were decreased,while G2/M phase were increased in U2OS,MG63 and SaOS2 cells,respectively.The results showed CB inhibited the proliferation of osteosarcoma cells through blocking the cell cycle in G2/M phase.Induction of apoptosis was confirmed by Hoechst 33258 and Annexin V/PI staining.After treating with 100 nmol/L CB for 48 h,the extents of apoptosis were 33.6％±6.4％,36.4％±7.8％ and 29.3％±5.1％,respectively.These results indicate that the anti-tumor activity of cinobufagin in osteosarcoma cells was due to a G2/M cell cycle arrest and apoptosis inducing effect.Western blot showed that CB could induce the apoptosis related family proteins Bax,cleaved-PARP up-regulation,xIAP,cIAP-1,survivin and p65 downregulation in OS cells.Conclusion CB can inhibit the cell viability and induce G2/M cell cycle arrest and apoptosis in U2OS,MG63 and SaO2 cells.The apoptosis-inducing effect of CB is confirmed by the regulation of apoptosis related proteins IAPs and Bcl-2 in vitro.
