冬凌草甲素诱导人卵巢癌SKOV3细胞凋亡及其机制

doi:10.3971/j.issn.1000-8578.2013.01.010

摘要: 目的观察中药冬凌草的有效提取成分冬凌草甲素对人卵巢癌细胞株SKOV3的增殖抑制及诱导凋亡作用，并探讨其可能的抗肿瘤作用机制。方法四甲基偶氮唑盐（MTT）比色法观察冬凌草甲素单药及冬凌草甲素联合顺铂对SKOV3细胞的增殖抑制情况；倒置显微镜和透射电子显微镜观察SKOV3细胞形态学和超微结构的改变；流式细胞术PI单染检测冬凌草甲素对细胞周期的影响；流式细胞术AnnexinV-FITC/PI双染检测冬凌草甲素对细胞凋亡的影响；实时荧光定量聚合酶连反应（QRT-PCP）检测SKOV3细胞bcl-2、bax、survivin mRNA 的表达水平。结果(1)MTT结果显示冬凌草甲素对卵巢癌SKOV3细胞的增殖具有明显的抑制作用，且呈时间剂量依赖性，24、48、72 h 的半数抑制浓度（IC₅₀）分别为45.22、33.85、22.48 μmol/L；与顺铂合用具有协同抑制作用。(2)倒置显微镜下可见细胞生长明显受抑制。(3)透射电子显微镜结果显示，冬凌草甲素组出现空泡变性，染色质浓缩，核染色质边集。(4)细胞周期显示冬凌草甲素组细胞周期被阻滞于S期和G₂／M期。(5)AnnexinV - FITC /PI双染检测冬凌草甲素（10、20、30 μmol/L）作用细胞48h后细胞的凋亡率分别为（9.13±0.91）%、（18.89±1.79）%、（36.11±3.01）%,明显高于阴性对照组，差异有统计学意义(P<0.05）。(6)QRT-PCR 显示冬凌草甲素组bcl-2、survivin基因表达下调，bax 基因表达上调，与阴性对照组相比差异有统计学意义(P<0.05）。结论冬凌草甲素对人卵巢癌SKOV3细胞具有明显的增殖抑制及诱导凋亡作用;其抗肿瘤机制可能与下调bcl-2、survivin,上调bax的表达有关。

Abstract: Objective To investigate the effect of oridonin which is extractsed from Donglingcao on the suppression of growth and induction of apoptosis in human ovarian cancer SKOV3 cell,and explore its mechanism. Methods The growth inhibitory effect of oridonin with or without cisplatin on SKOV3 cells was detected by MTT assay;The morphological and ultrastructural changes of SKOV3 cells were observed by inverted microscope and transmission electron microscope,respectively;The effect of cell cycle and apoptosis rate of oridion on SKOV3 cells were examined by Flow Cytometry (FCM);The expression of bcl-2,bax and survivin mRNA of SKOV3 cells was detected by quantitative reversed transcription-polymerase chain reaction (QRT-PCR). Results (1)MTT assay showed that oridonin could significantly inhibit the proliferation of SKOV3 cells in time and dose-dependent manner,and the half maximal inhibitory concentration (IC₅₀) of 24,48,72 hours were 45.22,33.85，22.48 μmol/L,respectively；the inhibitory effect of oridonin combined with cisplatin was more obvious than either one used separately.(2)Inverted microscope observed that cell growth was obviously inhibited.(3)Apoptosis morphological changes about vacuolar degeneration,chromatic agglutination and nuclear condensation was detected by electron microscope in oridonin-treated SKOV3 cells.(4)Cell cycle analysis indicated that the oridonin-treated cells were arrested at the S and G2/M phases.(5)The results of AnnexinV-FITC/PI double-stained represented the apoptosis rate of SKOV3 cells treated with 10,20 and 30 μmol/L oridonin for 48 hours were（9.13±0.91）%,（18.89±1.79）%,（36.11±3.01）%,respectively,and the apoptotic rate increased significantly compared with control group,the difference was statistically significant(P<0.05).(6)QRT-PCR showed that the expression of bcl-2 and survivin declined and bax upregulated in oridonin-treated SKOV3 cells,and the difference was statistically significant compared with control group (P<0.05). Conclusion Oridonin can inhibit the proliferation and induce the apoptosis of SKOV3 cells，and its mechanism might be related to up-regulate the expression of bax and down-regulate the expression of bcl-2 and survivin.

冬凌草甲素诱导人卵巢癌SKOV3细胞凋亡及其机制

Pro-apoptotic Effect and Its Mechanisms of Oridonin on Human Ovarian Cancer Cell SKOV3