冬凌草甲素对Nalm-6细胞的增殖抑制作用及其作用机制  

Anti-proliferation effect of oridonin on Nalm-6 cells and its mechanisms of action

作　　者：刘加军[1] 张俊峰[2] 林东军[1] 陆敏强[2] 

机构地区：[1]中山大学附属三院血液内科,广东广州510630 [2]中山大学附属三院肝移植中心,广东广州510630

出　　处：《中华肿瘤防治杂志》2009年第13期966-969,989,共5页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(30772782);教育部新世纪优秀人才支持计划资助项目(NCET-06-0721);广东省中医药局课题(2007103)

摘　　要：目的:探讨冬凌草甲素(oridonin,ORI)对淋巴细胞白血病Nal m-6细胞的增殖抑制作用及其作用机制。方法:以不同浓度的ORI(0～48μmol/L)作用于体外培养的Nal m-6细胞0、24、48及72 h,应用MTT法检测细胞生长抑制率,流式细胞仪检测细胞凋亡率,琼脂糖凝胶电泳观察细胞凋亡时的DNA梯状条带,应用透射电镜观察细胞形态学的变化,用蛋白质印迹法检测Caspase-3及其裂解底物多聚(ADP-核糖)聚合酶PARP[(poly(ADP-ribose)polymerase)]表达水平的变化,并对凋亡调节蛋白Bax及Bcl-2的表达水平进行检测。结果:>16μmol/L的ORI可显著抑制细胞的生长及诱导细胞发生凋亡,呈现出明显的量-效与时-效关系,药物作用48 h后在琼脂糖凝胶电泳上可见明显的DNA梯状条带。蛋白质印迹法检测结果表明,Caspase-3被活化出现相对分子质量20×103的亚单位,同时PARP被裂解出现相对分子质量89×103的亚单位片段。同时,药物作用48 h后凋亡抑制蛋白Bcl-2的表达水平明显降低,而促凋亡蛋白Bax的表达水平显著升高。结论:ORI能显著抑制Nal m-6细胞的生长并诱导细胞发生凋亡,通过激活Caspase-3以及降低Bcl-2、升高Bax的表达水平是ORI诱导细胞发生凋亡的重要作用机制;这些结果表明ORI是一种潜在的抗白血病药物。OBJECTIVE： To investigate the anti-proliferation effect of oridonin on leukemic Nalm-6 cells and its mechanisms of action. METHODS： Nalm-6 cells in culture medium in vitro were treated by oridonin in different concentrations （0-48 μmol/L） for 0,24, 48 and 72 h. The inhibitory rates of the ceils were measured by MTT assay, cell apoptotie rates were detected by flow cytometry （FCM）, DNA fragmentation was as sayed by agarose gel electrophoresis, cell morphology was observed by an electron microscope, and Bcl-2 and bax as well as Caspase-3 and poly （ADP ribose） polymerase （PARP） expres sions were detected by Western blotting. RESULTS： Oridonin （over 16 μmol/L） could inhibit the growth of Nalm-6 cells and cause apoptosis remarkably, and the suppression was both in a time and dose-dependent manner. DNA ladder was observed af ter the cells treated by oridonin for 48 h, and marked morphological changes of cell apoptosis such as condensation of chroma tin were clearly observed by an electron microscope. Western blotting showed the cleavage of the Caspase-3 zymogen protein, with the appearance of its 20 × 10^3 subunit, and a cleaved 89 × 10^3 fragment was also found. Furthermore, Western blotting al so showed that anti apoptotic protein Bcl-2 was decreased re- markably while pro-apoptotic protein bax increased significantly after the cells were treated by oridonin for 48 h. CONCLUSIONS： Oridonin can inhibit cell growth and induce apoptosis in Nalm-6 cells via activation of Caspase-3 as well as downregulation of Bcl 2 and upregulation of bax expression of Nalm-6 cells. The resuhs indicate that oridonin may be an important potential anti-leukaemia reagent.

关 键 词：冬凌草素/药理学 白血病 淋巴细胞 细胞凋亡 细胞增殖 

分 类 号：R285[医药卫生—中药学]