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目的研究渥曼青霉素(Wortmannin)联合丝裂霉素C(mitomycin C,MMC)对肝癌HepG-2细胞增殖、凋亡及相关基因Cyclin D1、Bad表达的影响。方法实验分4组,对照组:未处理的HepG-2细胞;Wortmannin组:用终浓度为25、50、100、200、400nmol/L Wortmannin处理的HepG-2细胞;MMC组:用终浓度为1.875、3.75、7.5、15、30nmol/LMMC处理的HepG-2细胞;联合用药组:同时用Wortmannin和MMC处理HepG-2细胞,终浓度为26.875、53.75、107.5、215、430nmol/L。MTT比色法检测细胞增殖状态;Hoechst 33258荧光染色观察凋亡细胞核形态学变化,流式细胞术检测肿瘤细胞周期和凋亡;逆转录聚合酶联反应及Western blot检测PI3K/Akt信号传导通路相关基因和蛋白的表达。结果联合用药组的细胞增殖抑制率显著高于单独用药组(P<0.05);联合用药作用24h后流式细胞术显示细胞明显阻滞于G0/G1期;细胞凋亡率由处理前的(3.23±1.32)%提高到(14.82±1.36)%(P<0.01);荧光显微镜下可见典型的细胞凋亡形态学改变;联合用药后细胞p-Akt活化程度降低,Cyclin D1表达量减少,Bad表达量增加(P<0.05)。结论渥曼青霉素与丝裂霉素联合应用,具有协同抗肿瘤效应。
Objective To investigate the effects of Wortmannin combined with mitomycin C on the proliferation and apoptosis of hepatocellular carcinoma HepG-2 cells and the expression of Cyclin D1 and Bad genes. Methods The experiment was divided into 4 groups, control group: untreated HepG-2 cells; Wortmannin group: HepG-2 cells treated with Wortmannin at a final concentration of 25, 50, 100, 200 and 400 nmol / L; MMC group: HepG-2 cells treated with 1.875, 3.75, 7.5, 15, 30 nmol / LMMC; Combination group: HepG-2 cells were treated with Wortmannin and MMC at the final concentration of 26.875,53.75,107.5,215,430nmol / L. MTT assay was used to detect the cell proliferation status; Hoechst 33258 fluorescence staining was used to observe the morphological changes of apoptotic cells; flow cytometry was used to detect cell cycle and apoptosis; RT-PCR and Western blot were used to detect the PI3K / Akt signaling pathway Gene and protein expression. Results The inhibitory rate of cell proliferation in the combination group was significantly higher than that in the drug alone group (P <0.05). Flow cytometry showed that the cells were arrested in the G0 / G1 phase after 24 h combined treatment. 3.23 ± 1.32)% increased to (14.82 ± 1.36)% (P <0.01). The morphological changes of typical apoptotic cells were observed under fluorescence microscope. The activation of p-Akt decreased, the expression of Cyclin D1 decreased, Bad The expression level increased (P <0.05). Conclusions Wortmannin combined with mitomycin has a synergistic anti-tumor effect.