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目的:探讨黏液瘤病毒(myxomavirus,MV)感染对人卵巢癌SKOV3细胞增殖的影响及其分子机制。方法:体外培养人卵巢癌SKOV3细胞,给予MV感染处理;同时,以灭活病毒处理作为阴性对照组,以RPMI 1640培养液处理作为空白对照组。采用CCK-8法检测各组卵巢癌SKOV3细胞的增殖情况,实时荧光定量PCR法检测各组细胞中Bcl-2和survivin的m RNA水平,FCM法分析各组细胞周期分布,蛋白质印迹法检测各组细胞中细胞外调节蛋白激酶1/(2extracellular regulated protein kinase 1/2,ERK1/2)、蛋白激酶B(protein kinase B,PKB,又称Akt)、磷酸化ERK1/2、磷酸化Akt、Bcl-2和survivin的蛋白表达水平,并采用比色法测定caspase-3和caspase-8的活性。结果:与阴性对照组和空白对照组相比,MV能够明显抑制人卵巢癌SKOV3细胞的增殖和细胞周期进程(P值均<0.05)。MV作用SKOV3细胞96 h后,抗凋亡蛋白Bcl-2和survivin的m RNA及蛋白表达水平均明显下调(P值均<0.05),增殖相关信号通路中ERK1/2和Akt的磷酸化水平均明显降低(P值均<0.05),而caspase-3和caspase-8的活性均明显升高(P值均<0.05)。结论 :MV能明显抑制卵巢癌细胞的增殖,其作用机制可能与阻滞细胞周期,下调抗凋亡蛋白Bcl-2和survivin的表达,提高caspase-3和caspase-8的活性,并抑制增殖相关信号通路中ERK1/2和Akt的磷酸化有关。
Objective: To investigate the effect of myxomavirus (MV) infection on the proliferation of human ovarian cancer cell line SKOV3 and its molecular mechanism. Methods: Human ovarian cancer SKOV3 cells were cultured in vitro and treated with MV. Inactivated virus was used as negative control and RPMI 1640 medium was used as blank control. The proliferation of ovarian cancer SKOV3 cells in each group was detected by CCK-8 method. The m RNA levels of Bcl-2 and survivin in each group were detected by real-time fluorescence quantitative PCR. The cell cycle distribution was analyzed by FCM, The expression of extracellular regulated protein kinase 1/2, PKB (Akt), phosphorylated ERK1 / 2, Akt, Bcl -2 and survivin, and the activity of caspase-3 and caspase-8 were determined by colorimetry. Results: Compared with the negative control group and the blank control group, MV significantly inhibited the proliferation and cell cycle progression of human ovarian cancer SKOV3 cells (all P <0.05). The expression of m RNA and protein of anti-apoptotic proteins Bcl-2 and survivin were significantly down-regulated in SKOV3 cells (all P <0.05) after 96 h of MV treatment. The phosphorylation levels of ERK1 / 2 and Akt (P <0.05), while the activities of caspase-3 and caspase-8 were significantly increased (P <0.05). Conclusions: MV can significantly inhibit the proliferation of ovarian cancer cells. The mechanism may be related to arresting cell cycle, down-regulating the expression of anti-apoptotic proteins Bcl-2 and survivin, increasing the activity of caspase-3 and caspase-8 and inhibiting the proliferation ERK1 / 2 and Akt phosphorylation in the signaling pathway.