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目的 :观察阿法替尼(afatinib)联合Mithramycin A(MIT)对人肝癌Hep G2细胞增殖、凋亡的作用以及相关因子表达的影响。方法:将afatinib与MIT单独或联合作用于肝癌Hep G2细胞,采用MTT法测定药物对细胞生长的抑制率,并运用倒置显微镜观察药物作用后细胞形态学变化;以流式细胞技术测定药物对细胞周期和凋亡的影响;以实时荧光定量聚合酶链反应(q RT-PCR)定量测定细胞内表皮生长因子受体(EGFR)、Sp1、Sp3以及增殖、凋亡相关因子Cyclin-D1、Cyclin-E2、Bcl-2、Caspase3、Caspase9和p53的表达变化。结果 :afatinib与MIT均能有效抑制肝癌Hep G2细胞的生长,并且呈现时间依赖性,两药联合作用能明显增加抑制率(P均<0.05);联合用药48 h后,可诱导Hep G2细胞产生G0/G1期阻滞并诱发凋亡,抑制作用及凋亡率均较单药组增高(P均<0.05);另外,给药72 h后,单药组均出现不同程度的Cyclin-D1、Cyclin-E2、Bcl-2 m RNA表达量下降,并伴有Caspase3基因上调。单用afatinib组同时出现Caspase9和p53的表达上调,MIT组检测到EGFR、Sp1和Sp3的同步减少,联合用药组以上改变较单药组明显(P均<0.05)。结论:afatinib联合MIT能有效抑制肝癌Hep G2细胞增殖、促进凋亡,这可能与药物作用后Cyclin-D1、Cyclin-E2、Bcl-2下调以及Caspase3、Caspase9和p53的表达上调相关。此项研究可能为以EGFR为中心的肝癌联合治疗提供新方向。
Objective: To observe the effects of afatinib combined with Mithramycin A (MIT) on the proliferation and apoptosis of Hep G2 cells and the expression of related factors. Methods: The effect of afatinib and MIT alone or in combination on Hep G2 cells was determined by MTT method. The morphological changes of the cells were observed by inverted microscope. The effect of drug on cells Cyclin-D1 and Cyclin-D1 were detected by real-time fluorescence quantitative polymerase chain reaction (q RT-PCR) .Results The levels of epidermal growth factor receptor E2, Bcl-2, Caspase3, Caspase9 and p53 expression changes. Results: Both afatinib and MIT could effectively inhibit the growth of Hep G2 cells in a time-dependent manner. The combination of both afatinib and MIT significantly increased the inhibition rate (all P <0.05). After 48 h, Hep G2 cells could be induced In the G0 / G1 phase, the apoptosis and apoptosis were inhibited and the apoptosis rate was higher than that in the monotherapy group (all P <0.05). In addition, the levels of Cyclin-D1, Cyclin-E2, Bcl-2 m RNA expression decreased, accompanied by Caspase3 gene upregulation. The expression of Caspase9 and p53 was up-regulated in afatinib alone group. The synchronous decrease of EGFR, Sp1 and Sp3 was detected in MIT group. The change in combined group was more obvious than that in single drug group (all P <0.05). Conclusions: Afatinib combined with MIT can effectively inhibit Hep G2 cell proliferation and promote apoptosis, which may be related to down-regulation of Cyclin-D1, Cyclin-E2, Bcl-2 and up-regulation of Caspase3, Caspase9 and p53. This study may provide a new direction for the combined treatment of liver cancer with EGFR as the center.