大蒜素对脑胶质瘤C6细胞增殖和凋亡的实验研究  

Experimental Study of Allicin on Proliferation and Apoptosis of Brain Glioma C6 Cells

作　　者：方伟[1] 陈景南[1] 范成谱 杜杭根[2] 

机构地区：[1]浙江中医药大学第二临床医学院,杭州310053 [2]浙江中医药大学附属第二医院

出　　处：《浙江中医药大学学报》2016年第10期762-767,共6页Journal of Zhejiang Chinese Medical University

基　　金：浙江省中医药科技计划项目(2015ZA106)~~

摘　　要：[目的]探讨大蒜素对体外培养的脑胶质瘤C6细胞增殖抑制和诱导凋亡的作用及其机制。[方法]培养脑胶质瘤C6细胞至对数生长期,不同浓度大蒜素作用下,于倒置显微镜及Hoechst33258荧光染色显微镜下观察C6细胞形态学变化;四甲基偶氮噻唑蓝法(tetrazolium salt colorimetry,MTT)检测不同浓度大蒜素对C6细胞增殖的影响并计算抑制率;钙依赖性磷脂结合蛋白-荧光素/碘化丙啶双染法(Annexin V-fluorescein/propidium iodide,Annexin V-FITC/PI)检测凋亡率;流式细胞术(flow cytometry,FCM)检测细胞周期;蛋白质印迹法(western blotting,WB)检测Bax、Bcl-2基因的表达。[结果]大蒜素作用C6细胞24h后,于倒置显微镜观察可见C6细胞生长明显抑制,部分细胞皱缩变形,出现成泡现象;Hoechst33258荧光染色显微镜下观察发现凋亡的细胞,细胞的体积变大,核固缩、凝集,核染色质边集,出现浓染致密的颗粒块荧光信号,并随着大蒜素浓度增加表现越加明显。不同浓度(5、10、20、40、80μg·m L^(-1))大蒜素作用C6细胞(24h、48h、72h)后,细胞增殖受到抑制,与0μg·m L^(-1)大蒜素比较差异有统计学意义(P<0.01);同一浓度大蒜素作用C6细胞48h、72h后,与24h比,差异有统计学意义(P<0.01)。随着大蒜素浓度的增加,C6细胞凋亡率上升,与0μg·m L^(-1)大蒜素比较差异有统计学意义(P<0.01);其中80μg·m L^(-1)大蒜素显著抑制C6细胞增殖、诱导其凋亡,与各浓度大蒜素比,差异有统计学意义(P<0.01)。20μg·m L^(-1)大蒜素作用C6细胞24h后,与0μg·m L^(-1)大蒜素比G0/G1期细胞比例增加,S、G2/M期细胞比例降低,差异有统计学意义(P<0.05)。随着大蒜素浓度的增加Bax表达水平上升、Bcl-2表达水平下降,Bcl-2/Bax比值逐渐上升,与0μg·m L^(-1)大蒜素比较差异有统计学意义(P<0.01);其中80μg·m L^(-1)大蒜素与5、20μg·m L^(-1)大蒜素比,差异有统计学意义(P<0.01)。[结论]大蒜素能体外抑制C6细胞的�[Objective]To explore the effect of allicin in vitro cultured brain glioma C6 cells inhibit proliferation and induce apoptosis and its mechanism.[Methods]Different concentrations of allicin action C6 glioma cells that were cultured to logarithmic growth phase,C6 cell morphological changes was observed under inverted microscope and Hoechst33258 fluorescence staining microscope;MTT assays was used to detect different concentrations of allicin on C6 cells proliferation and calculate the inhibition rate;Annexin V-FITC/PI double staining and FCM was used to detect apoptosis rate and cell cycle,respectively; Western blotting was used to detect Bax and Bcl-2 gene expression.[Results]Allicin on C6 cells after 24 hours, inverted microscope shows the growth of C6 cells significantly inhibited, part of the cell shrinkage deformation and into a bubble;Hoechst33258 fluorescent staining microscope reveals that the apoptosis of cells,cell volume gets bigger, nuclear condensation, aggregation,nuclear staining set qualitative change occurs stain dense particles block the fluorescence signal and the increased concentration of allicin more obvious manifestations. Different concentrations（5, 10, 20, 40, 80 μg·m L^-1） allicin on C6cells（24h,48 h,72h） reveal proliferation is significantly suppressed,the differences are statistiaclly significant compare with 0 μg·m L^-1allicin（P〈0.01）;same concentration of allicin on C6 after 48 h, 72 h,the differences are statistiaclly significant compared with 24h（ P〈0.01）.With the increase of concentration of allicin,C6 cells apoptosis rate increases significantly, the difference is statistically significant compared with 0 μg·m L^-1allicin（P〈0.01）.The 80μg·m L^-1allicin significantly inhibits C6 cell proliferation and induces apoptosis compared with each concentration of allicin, the difference is statistically significant（ P〈0.01）.20μg·m L^-1allicin on C6 cells after 24 h, compared with 0μg·m L^-1allicin in G0/G1 phase cell proportion increases,S,G2/M ph

关 键 词：大蒜素 胶质瘤C6细胞 增殖 凋亡 BCL-2 BAX 

分 类 号：R331[医药卫生—人体生理学]