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目的:探讨丙戊酸钠(valproate acid,VPA)联合全反式维甲酸(all-trans retinoic acid,ATRA)对子宫颈癌HeLa细胞的杀伤作用及其可能机制。方法:采用3 mmol/L VPA、1μmol/L ATRA、1μg/mL顺铂(cis-diamminedichloroplatinum,DDP)单独或联合作用于子宫颈癌HeLa细胞,对照组仅加溶媒;采用MTT法检测细胞生长抑制率;Hochest33258标记法观察细胞凋亡情况;RT-PCR法检测凋亡相关基因p53、信号转导及转录激活因子(signal transducers and activators of transcription,STAT3)、p53上调凋亡调控因子(p53 up-regulated modulator of apoptosis,PUMA)及Bax、Bcl-2和Bcl-XL的mRNA表达;Western印迹法检测p53、STAT3及磷酸化STAT3(phosphorylated STAT,p-STAT3)蛋白表达的变化。结果:VPA对子宫颈癌HeLa细胞有生长抑制作用,且VPA联合ATRA优于单独用药。Hochest33258标记发现,用药后细胞出现核碎裂等典型的凋亡改变,VPA+ATRA组细胞凋亡率显著高于对照组。RT-PCR结果显示,VPA可在mRNA水平上增强p53、PUMA及Bax的表达,减弱STAT3的表达,且联合用药组效果更明显,并呈时间依赖关系;而Bcl-XL及Bcl-2的表达无明显变化。Western印迹结果显示,VPA联合ATRA显著增强子宫颈癌HeLa细胞中p53蛋白表达,降低STAT3和p-STAT3蛋白表达水平,亦呈现明显的时效关系。结论:VPA单独及联合ATRA在体外可有效杀伤子宫颈癌HeLa细胞株。推测其可能的作用机制是诱导p53表达水平上调,STAT3及p-STAT3表达水平下调。
Objective: To investigate the killing effect of valproate acid (VPA) combined with all-trans retinoic acid (ATRA) on cervical cancer HeLa cells and its possible mechanism. Methods: Cervical cancer HeLa cells were treated with 3 mmol / L VPA, 1 μmol / L ATRA and 1 μg / mL cisplatin (DDP) alone or in combination. The control group was given only vehicle. The cell growth inhibition Hochest33258 labeling method was used to observe the cell apoptosis. RT-PCR was used to detect the expression of p53, signal transducers and activators of transcription (STAT3), p53 up- regulated modulator of apoptosis (PUMA), Bax, Bcl-2 and Bcl-XL mRNA expression. Western blotting was used to detect the protein expression of p53, STAT3 and phosphorylated STAT3. Results: VPA could inhibit the growth of cervical cancer HeLa cells, and VPA combined with ATRA was better than single drug. Hochest33258 labeling found that the typical apoptotic changes such as nuclear fragmentation occurred after treatment. The apoptosis rate of VPA + ATRA group was significantly higher than that of control group. The results of RT-PCR showed that VPA could enhance the expression of p53, PUMA and Bax at the mRNA level and weaken the expression of STAT3, and the combined effect was more obvious and time-dependent; while the expression of Bcl-XL and Bcl-2 No significant changes. Western blotting results showed that VPA combined with ATRA significantly increased p53 protein expression in cervical cancer HeLa cells and decreased STAT3 and p-STAT3 protein expression, also showed a significant aging relationship. Conclusion: VPA alone and in combination with ATRA can effectively kill cervical cancer HeLa cell line in vitro. It is speculated that its possible mechanism of action is to induce the up-regulation of p53 expression and the down-regulation of STAT3 and p-STAT3 expression.