联合应用干扰素α-2b及选择性环氧化酶2抑制剂对肝癌细胞抑制作用的研究

摘要

目的 观察干扰素α-2b(IFN-α-2b),选择性环氧化酶2(COX-2)抑制剂非甾体类消炎药塞来昔布单用以及两者联合应用时,对人肝癌细胞株HepG2的生长抑制作用及同时检测药物作用后COX-2和血管内皮生长因子(VEGF)及Bcl-2表达的情况,探讨药物作用的机制.方法 HepG2细胞根据所加药物不同分为不同浓度的IFN-α-2b组(1250、2500、5000、10 000、20 000、40 000 U/mL)、塞来昔布组(25、50、100、200 μmol/L)、联合用药组(IFN-α-2b 5000 U/mL+塞来昔布 50 μmol/L、IFN-α-2b 5000 U/mL+选择性COX-2 100 μmol/L).MTT法检测不同时间(24、48 h)各组细胞增殖抑制率,流式细胞术检测细胞凋亡率,Western blot检测用药前后COX-2、VEGF、Bcl-2蛋白在HepG2细胞中的表达变化.结果 不同浓度的塞来昔布和干扰素对HepG2细胞均有抑制作用,且联合效果明显优于单用,各组与对照组差异有统计学意义(P＜0.05);流式细胞术分析:塞来昔布50 μmol/L组、塞来昔布100 μmol/L组、IFN-α-2b 5000 U/mL组、IFN-α-2b 5000 U/mL+塞来昔布50 μmol/L组、IFN-α-2b 5000 U/mL+塞来昔布100 μmol/L组细胞凋亡率分别为(14.93±0.79)%、(25.43±0.89)%、(20.52±1.46)%、(27.12±3.34)%、(48.17±2.04)%,与阴性对照组(8.73±0.83)%比较差异均有统计学意义(P＜0.05);塞来昔布单用及联合IFN-α-2b作用48 h后,各组COX-2、VEGF、Bcl-2蛋白的表达下调,呈剂量依赖性,联合用药组较单药组作用明显.结论 干扰素α-2b和塞来昔布均有抑制肝癌细胞HepG2增殖的作用,同时对细胞具有凋亡诱导作用,亦能抑制细胞中COX-2、VEGF、Bcl-2的表达.上述抑制作用随药物浓度的增加、作用时间的延长而增强,联合作用更强.%Objective To investigate the growth inhibition and its molecular mechanism on human hepatoma cell line HepG2 through application of celecoxib, cyclooxygenase-2 inhibitor, interferon alfa-2b alone or in combination with each other,and test the expression of the cyclooxygenase-2( COX-2 /vascular endothelial growth factor ( VEGF )and Bcl-2 To explore the mechanism in inhibition of proliferation of HepG2 cells. Methods The cells of human hepatoma HepG2 were divided into 1250,2500,5000,10000,20000 and 40000 U/mL IFN-a-2b groups, 25,50,100,200 μmol/L celecoxib groups and IFN-a-2b 5000 U/mL + celecoxib50 μmol/L, IFN-a-2b 5000 U/ mL + celecoxib 100 μmol/L combination groups. The inhibitory rates were detected by MTT assay after HepG2 were treated for 24 and 48h. Analyze the apoptosis of the tumor cells by flow cytometry. Detect the expression of COX-2, VEGF and Bcl-2 by Western blot assay. Results The inhibiton of IFN-a-2b and celecoxib when applicat-ed on the proliferation of HepG2 cells:After treated with IFN-a-2b and celecoxib respectively or in combination for 24h and 48h,cells proliferation were obviously inhibited,and the role in combined treatment groups was more significant than monotherapy groups compared with the negative control group( P < 0. 05 ). Flowcytometry showed that:The apoptosis rate of celecoxib 50 μmol/L group,celecoxib 50 μmol/L group,IFN-a-2b 5000 U/mL group, IFN-a-2b 5000 U/mL + celecoxib 50 μmol/L group and IFN-a-2b 5000 U/mL + celecoxib 100 μmol/L group were respectively compared with the negative control group( P < 0. 05 ). Western-blot assay results showed that 48 hours after IFN-a-2b and celecoxib administration, the expression of COX-2, VEGF, Bcl-2 and gradually decreased , and the role in combined treatment groups was more significant than monotherapy groups. Conclusion IFN-a-2b and celecoxib have inhibitory effects on the growth of human hepatoma cell line HepG2 ,and they can inhibit the expressions of COX-2 , VEGF and Bcl-2 in HepG2 cells in a dose and time dependent manner.