葡萄籽提取物诱导人卵巢癌SKOV3细胞凋亡及其机制的探讨

Apoptosis-inducing effect of grape seed proanthocyanidin extract on human ovary cancer SKOV3 cells and its mechanism

目的:探讨葡萄籽提取物(Grape seed extract,GSE)对卵巢癌SKOV3细胞增殖和凋亡的影响及其机制。方法:体外培养的SKOV3细胞与25、50、100、200、400μg/ml的GSE共孵育,分别在24、48和72h时观察细胞形态学变化,MTT检测细胞增殖抑制率,流式细胞仪和Tunel分析细胞凋亡,Western-blot检测SKOV3细胞Caspase-3在蛋白水平的表达。结果:不同浓度(25、50、100、200、400μg/ml)GSESKOV3细胞作用48h的细胞增殖抑制率分别为48.67%、53.85%、67.03%、73.78%和77.39%。流式细胞和TUNEL检测经25μg/mlGSE处理24、48h后,SKOV3细胞凋亡率分别为:31.98%、45.78%。GSE还可以明显增强Caspase-3蛋白的表达水平。这些作用均随GSE浓度和作用时间的延长而增强。结论:GSE可在体外抑制SKOV3细胞增殖,诱导其凋亡。其机制可能与激活Caspase途径有关。

Objective： To study the biological effects of grape seed extract （ GSE ）on the human ovary cancer cells and explore its molecular mechanism. Methods： Human ovary cancer cells line SKOV3 were cultured.They were put into 96-well plate and added with solutions of GSE of the terminal concentrations of 25,50,100,200,400 μ g/ml respectively, and 24,48,72 hours later cell growth curve was drawn. MTF assay was used for other SKOV3 cells co-incubated with GSE of the terminal concentration of 25,50,100,200,400 μg/ml respectively so as to calculate the proliferation inhibition rate. SKOV3 cells were treated with GSE of the concentration of 25 μ g/ml for 24,48 hours respectively. Flowcytometry （FCM）was used to analyze the DNA cycle and TUNEL was used to calculate the apoptotic rate. Annexin-V labeling method was used to detect the positive rate of apoptotic cells. SKOV3 ceils were treated with GSE of the concentration of 25 μg/ml for 24,48,72 hours.Western blotting was used to detect the protein expression of caspase-3. Results：GSE dose-dependently inhibited the proliferation of the SKOV3 cells in a dose-dependent manner.Treated by GSE, the progress of cells at stage into GJM stage was inhibited.Tunel showed that treated by GSE （25 μ g/ml） for 24,48 h the apoptotic rates of the cells were 31.98% and 45.78% , respectively. Annexin-V showed that after incubation with GSE （25 μ g/ml） for 12,24,48 h, the apoptotic rate were 6.71%, 19.05%, 36.55% ,respectively. Western blotting showed that the caspase-3 protein expression were up-regulated.Conclusion：GSE inhibits the proliferation of malignant human ovary cancer cells and induces their apoptosis..Expression of caspase-3 protein may be related to cell growth inhibition and the apoptosis mediated by GSE in vitro.