摘要
目的探讨去甲斑蝥素(NCTD)对微管蛋白聚合-解聚过程的影响。方法采用蛋白凝胶电泳鉴定从猪脑中提取的微管蛋白,并用Bradford法测定微管蛋白含量。在经过秋水仙碱和紫杉醇验证稳定的37℃和4℃平衡体系内加入NCTD 10,20和30μmol.L-1观察微管蛋白聚合-解聚活性。人卵巢癌SK-OV-3细胞加入NCTD 60μmol.L-1作用8 h,取上清和沉淀,Western印迹法检测微管蛋白含量。结果与空白对照组相比,在37℃聚合反应中,NCTD 10,20和30μmol.L-1组对微管蛋白聚合的抑制率分别为(5.5±2.7)%,(7.1±1.2)%和(27.5±0.4)%,在此浓度范围内NCTD随浓度的升高抑制作用增强(P<0.05),但对微管蛋白在4℃的解聚影响不显著。NCTD 60μmol.L-1作用人卵巢癌细胞后,细胞中游离微管蛋白增加了(91.5±8.5)%,聚合微管蛋白量减少了(51.8±3.8)%,说明NCTD抑制了人卵巢癌细胞SK-OV-3中微管蛋白的聚合。结论 NCTD对体外微管蛋白的聚合具有一定的抑制作用。
OBJECTIVE To investigate the effect of norcantharidin（NCTD） on the polymerization-depolymerization process of tubulin in vitro.METHODS Microtubule was isolated from porcine brain and identified by SDS-PAGE.The concentration of tubulin was determined by Bradford method.After NCTD at concentrations of 10,20 and 30 μmol·L-1 was added into the tubulin polymerization-depolymerization system in vitro,the absorbance changes were measured under 350 nm at 37℃ and 4℃ respectively.Human ovarian tumor SK-OV-3 cells were used to analyze the effect of NCTD on tubulin polymerization-depolymerization at a cellular level.Microtubulin in supernatant and precipitation of SK-OV-3 cells was detected respectively by Western blotting after being treated by NCTD 60 μmol·L-1 for 8 h.RESULTS Tubulin polymerization was inhibited by NCTD in a concentration-dependent manner in 0~30 μmol·L-1 at 37℃.The inhibitory rate treated by different concentrations of NCTD in 10,20 and 30 μmol·L-1 was（5.5±2.7）%,（7.1±1.2）% and（27.5±0.4）%,respectively.However,the effect of NCTD on tubulin depolymerization at 4℃ was in significant.NCTD 60 μmol·L-1 increased free tubulin（91.5±8.5）% and decreased combinative tubulin（51.8±3.8）% in human ovarian cancer SK-OV-3 cells,suggesting that NCTD can inhibit tubulin polymerization at a cellular level.CONCLUSION NCTD can inhibit tubulin polymerization in vitro.
出处
《中国药理学与毒理学杂志》
CAS
CSCD
北大核心
2012年第5期630-634,共5页
Chinese Journal of Pharmacology and Toxicology
基金
北京中医药大学自主选题项目资助(2009JYBZZ-JS038)
北京中医药大学自主选题项目资助(2011JYBZZ-XS037)
北京中医药大学自主选题项目资助(2011JYBZZ-XS054)~~
作者简介
洪兴福（1984-），男，硕士研究生；
孙震晓（1967-），女，教授，博士，主要从事中药及其有效成分抗肿瘤分子细胞药理学研究。
