摘要
目的考察去甲斑蝥素(NCTD)对人卵巢癌SK-OV-3细胞生长的抑制作用,探究其诱导细胞发生有丝分裂期阻滞及凋亡的过程及相关性。方法 NCTD 30,60,120和240μmol.L-1分别作用人卵巢SK-OV-3细胞24,48和72 h后,MTT法检测细胞存活率;NCTD 60μmo.lL-1分别作用SK-OV-3细胞0,6,12和24 h后,流式细胞术测定细胞周期的变化;NCTD 60μmol.L-1分别作用SK-OV-3细胞0,6,12和24 h,倒置显微镜下检测其对细胞形态学变化的影响;Giemsa染色检测细胞核的变化;间接免疫荧光术结合激光扫描共聚焦显微镜技术检测细胞内微管分布及有丝分裂期纺锤体形成的影响。NCTD 60μmol.L-1分别作用12和36 h后,流式细胞术检测细胞凋亡情况;再将NCTD 60μmo.lL-1作用12 h后的细胞,采用温和的机械振荡法分离为悬浮细胞和贴壁细胞,继续作用24 h,流式细胞术及Giemsa染色检测分析两个时相两种细胞凋亡的影响。结果 MTT结果显示,NCTD 30～240μmol.L-1对SK-OV-3细胞的生长抑制作用存在明显的时间(P<0.05)和浓度依赖性(P<0.05);NCTD作用SK-OV-3细胞24,48和72 h的IC50分别为(261.3±2.4)μmo.lL-1,(48.3±1.7)μmol.L-1和(10.9±1.0)μmol.L-1。NCTD 60μmol.L-1分别作用于SK-OV-3细胞0,6,12和24 h,SK-OV-3细胞逐渐表现出G2/M期阻滞,呈时间依赖性;倒置相差显微镜下观察,NCTD引起SK-OV-3细胞逐渐收缩变圆,与周围细胞分离;Giemsa染色可见处于有丝分裂期的细胞显著增多,多个核细胞比例增多;免疫荧光染色发现,NCTD组细胞的微管系统受到干扰,有丝分裂期细胞纺锤体形成异常。NCTD作用细胞12和36 h凋亡率分别达20.4%和62.3%;将NCTD作用12 h的细胞,人工振荡分离为悬浮细胞和贴壁细胞,检测凋亡情况,同时检测继续作用24 h后两组细胞凋亡情况,发现处于有丝分裂间期的SK-OV-3细胞凋亡率大于处于有丝分裂期的细胞。结论 NCTD主要通过诱导人卵巢癌SK-OV-3细胞G2/M期阻滞和细胞凋亡抑制细胞生长。干扰�
OBJECTIVE To investigate the mechanisms of mitotic arrest and apoptosis of human ovarian cancer SK-OV-3 cells induced by norcantharidin （NCTD）. METHODS Cell viability was detec- ted by MTT assay after cells treated with NCTD 30, 60, 120 and 240 μmol· L-1 for 24, 48, and 72 h, respectively. Cell cycle and apoptosis were assayed through flow cytometry after cells treated with NCTD 60 μmol· L-1 for 0, 6, 12 and 24 h, respectively. Morphological changes in SK-OV-3 cells with NCTD 60 μmoI·L-1 treated for 0, 6, 12 and 24 h were observed under microscope, respectively. The morpho- logical changes in cell nuclei were observed after Giemsa staining, Effects of NCTD on microtubule organization and spindle formation in SK-OV-3 cells were detected and analyzed by indirect immunofluo- rescence staining and laser scanning confocal microscope. To investigate the relationship between apop- tosis and SK-OV-3 cells induced by NCTD 60μmoI·L-1 treated for 12 and 36 h, the cells were separated to adherent and non-adherent cells by gently physical shaking after NCTD was treated for 12 h and cells in 2 groups were continually treated with NCTD for 24 h, then analyzed by FCM and Giemsa staining. RESULTS Cell viability obviously decreased in NCTD 30 -240 μmOl·L-1 groups in a dose-dependent （ P 〈0.05） and time-dependent manner （ P 〈0, 05）. The IC50 of NCTD in SK-OV-3 cells treated for 24, 48 and 72 h were （261.3 ±2.4）μmoI·L-1 , （48.3±l.7）μmoI·L-1 and （10.9 ±l.0）μmoI·L-1 , respec- tively. After SK-OV-3 cells were treated with NCTD 60 μmol·L-1 for 0, 6, 12 and 24 h, G2/M phase cells accumulated in a time-dependent manner. Some cells showed cellular shrinkage and were separated from other cells and M phase cells increased in NCTD group. Immunostaining indicated that normal microtubule system of cells and mitotic spindle assembly were damaged in NCTD groups. In addition, the NCTD resulted in high cell apoptosis rate, which was 20.4% and 62.3% at 12 and 36 h. Analysis of apoptosis of adherent an
出处
《中国药理学与毒理学杂志》
CAS
CSCD
北大核心
2013年第2期180-186,共7页
Chinese Journal of Pharmacology and Toxicology
基金
北京中医药大学自主选题项目(2009JYBZZ-JS038)
北京中医药大学自主选题项目(2011JYBZZ-XS037)
北京中医药大学自主选题项目(2011JYBZZ-XS054)~~
