论文部分内容阅读
背景与目的:足叶乙甙(etoposide,VP鄄16)为白血病化疗最常用的化疗药之一,其临床应用日益受到天然与继发耐药的影响。对一些实体瘤的研究发现顺铂(cisplatin,DDP)与VP鄄16联合应用具有协同效应。本研究通过DDP与VP鄄16联合作用杀伤白血病细胞K562,探讨其增强VP鄄16疗效的作用机制。方法:采用VP鄄16(0,5μg/ml)与不同浓度DDP(0,0.3,3,15,30μg/ml)联合对K562细胞进行处理。应用噻唑蓝(MTT)法检测用药后细胞的存活相对数量,计算VP鄄16和DDP应用对K562细胞的抑制作用;AO/EB双荧光法定量分析细胞凋亡率。半定量RT鄄PCR法和Westernblot检测拓扑异构酶(topoisomerase,TOPO)Ⅱα、Ⅱβ、Sp1、Sp3基因的mRNA和蛋白水平。结果:VP鄄16与DDP合用具有明显的协同效应。两药合用后,细胞凋亡率明显增加。单独应用DDP可以明显上调TOPOⅡ和Sp1表达(呈浓度依赖,TOPOⅡα、Ⅱβ、Sp1在30μg/mlDDP时比正常对照分别上调36%,25%和75%),并使Sp3的表达降低49%;单用5μg/mlVP鄄16时TOPOⅡ则下调(TOPOⅡα下降72%),TOPOⅡβ和Sp1的表达未受影响,Sp3的表达上调14%。5μg/mlVP鄄16与DDP联合应用具有协同效应,使TOPOⅡ和Sp1表达水平升高。TOPOⅡα、Ⅱβ、Sp1在5μg/mlVP鄄16与30μg/mlDDP联合应用时比单用VP鄄16分别上升83%,11%,58%,但低于单独应用DDP的表达水平;而Sp3的表达水平与单独应用DDP比较下调61.3%。Western印迹检测结果与RT鄄PCR结果相符。结论:DDP在化疗中与VP鄄16发挥协同作用,通过上调拓扑异构酶Ⅱ的表达水平,为VP鄄16提供更多的靶酶,使VP鄄16杀伤K562细胞效果明显提高。
BACKGROUND & OBJECTIVE: Etoposide (VP-16) is one of the most commonly used chemotherapy drugs for leukemia chemotherapy. Its clinical application is increasingly influenced by natural and secondary drug resistance. The study of some solid tumors found that the combination of cisplatin (DDP) and VP-16 has a synergistic effect. In this study, DDP and VP-16 combined to kill leukemia cells K562, to explore its mechanism of enhancing the efficacy of VP-16. Methods: K562 cells were treated with VP-16 (0, 5μg / ml) and different concentrations of DDP (0,0.3,3,15,30μg / ml). Cell viability was measured by MTT assay. The inhibitory effect of VP-16 and DDP on K562 cells was calculated. AO / EB dual-fluorescence assay was used to quantify the apoptotic rate. Semi-quantitative RT-PCR and Western blot were used to detect the mRNA and protein levels of topoisomerase (TOPO) Ⅱα, Ⅱβ, Sp1 and Sp3 genes. Results: VP-16 combined with DDP had obvious synergistic effect. Combined use of two drugs, the apoptosis rate was significantly increased. TOPOⅡand Sp1were significantly upregulated by DDP alone (in a concentration-dependent manner, TOPOⅡα, Ⅱβand Sp1were up-regulated by 36%, 25% and 75% respectively at 30μg / ml DDP compared with the normal control), and the expression of Sp3 was reduced by 49% TOPOⅡ was down-regulated at 5μg / ml VP-16 (TOPOⅡα decreased by 72%). The expression of TOPOⅡβ and Sp1 was not affected, and the expression of Sp3 was up-regulated by 14%. The combination of 5μg / ml VP-16 with DDP has a synergistic effect, which makes the expression of TOPOⅡ and Sp1 elevated. TOPOⅡα, Ⅱβ and Sp1 were increased by 83%, 11% and 58%, respectively, compared with VP-16 alone at a dose of 5μg / ml VP-16 and 30μg / ml DDP but lower than that of DDP alone. Sp3 expression level Compared with the DDP alone application down 61.3%. Western blot results were consistent with RT-PCR results. CONCLUSION: DDP plays a synergistic role with VP-16 in chemotherapy. By up-regulating the expression of topoisomerase Ⅱ, DDP can provide more target enzymes for VP-16 and significantly reduce the effect of VP-16 on K562 cells.