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[目的]研究5-溴甲基-2(5H)呋喃酮诱导人肝癌HepG-2细胞株凋亡的作用机制.[方法]应用细胞体外传代培养方法培养人肝癌HepG-2细胞;利用四唑盐比色(MTT)法检测5-溴甲基-2(5H)呋喃酮对人肝癌HepG-2细胞株的抗增殖及细胞毒作用;采用流式细胞术检测癌细胞周期分布及药物对细胞凋亡的影响;采用免疫细胞化学技术检测5-溴甲基-2(5H)呋喃酮作用前后细胞凋亡相关基因Caspase-3,Caspase-8,Caspase-9,Survivin,Livin,NF-κB,Bcl-2,Bax,Fas蛋白的表达情况,并探讨其诱导人肝癌HepG-2细胞凋亡的作用机制.[结果]MTT检测结果显示,5-溴甲基-2(5H)呋喃酮对人肝癌HepG-2细胞的抑制作用表现出质量浓度和时间依赖性,IC50值为5 mg/L,48 h.流式细胞仪检测结果显示,5-溴甲基-2(5H)呋喃酮5 mg/L作用于人肝癌HepG-2细胞24,48,72 h后可见典型的亚二倍体峰,且G1期细胞数量增多,48 h时对照组G0/G1期细胞占40.30%,5-溴甲基-2(5H)呋喃酮组占46.21%,提示细胞阻滞在G0/G1期;对照组细胞凋亡率为0.02%,5-溴甲基-2(5H)呋喃酮组为19.74%,两组比较差异具有统计学意义(P<0.05).免疫细胞化学检测结果显示,与对照组比较,5-溴甲基-2(5H)呋喃酮组Survivin,Livin,NF-κB,Bcl-2蛋白的阳性表达率明显减少(P<0.05),Caspase-3,Caspase-8,Caspase-9,Fas及Bax蛋白的阳性表达率明显增加(P<0.05).[结论]5-溴甲基-2(5H)呋喃酮对人肝癌HepG-2细胞株具有明显的抑制增殖作用,且呈量-效和时-效关系,并将细胞阻滞于G0/G1期.5-溴甲基-2(5H)呋喃酮对人肝癌HepG-2细胞株有明显的诱导凋亡作用,作用机制可能是通过死亡受体途径和线粒体途径共同介导的.
[Objective] To investigate the mechanism of apoptosis of HepG-2 cells induced by 5-bromomethyl-2 (5H) furanone. [Methods] HepG-2 cells were cultured by passage in vitro. The antiproliferation and cytotoxicity of 5-bromomethyl-2 (5H) furanone on HepG-2 cells were detected by salt-colorimetric assay (MTT). The cell cycle distribution and drug- The apoptosis-related genes Caspase-3, Caspase-8, Caspase-9, Survivin, Livin and NF-κB were detected by immunocytochemistry before and after 5-bromomethyl- Bcl-2, Bax and Fas proteins in human hepatocellular carcinoma cell line HepG-2 were detected by MTT assay. [Results] The results of MTT assay showed that 5-bromomethyl-2 (5H) The inhibitory effect of HepG-2 cells in HepG-2 cells was mass-concentration-dependent and the IC50 was 5 mg / L for 48 h. The results of flow cytometry showed that 5-bromomethyl-2 (5H) / L on HepG-2 cells after 24,48,72 h showed a typical sub-diploid peak, and the number of cells in G1 phase increased, the control group G0 / G1 phase cells accounted for 40.30% 48 hours, 5-bromo Methyl-2 (5H) furan Group accounted for 46.21%, suggesting cell arrest in G0 / G1 phase; control group apoptosis rate was 0.02%, 5-bromomethyl-2 (5H) furanone group was 19.74%, the difference between the two groups was statistically significant (P <0.05) .The results of immunocytochemistry showed that the positive rates of Survivin, Livin, NF-κB and Bcl-2 in 5-bromomethyl-2 (5H) furanone group were significantly lower than those in the control group (P <0.05). The positive rate of Caspase-3, Caspase-8, Caspase-9, Fas and Bax protein increased significantly The hepatoma HepG-2 cell line had a significant inhibitory effect on proliferation, and showed a dose-effect and time-effect relationship, and arrested cells in the G0 / G1 phase. The inhibitory effect of 5-bromomethyl-2 (5H) HepG-2 cell line has obvious apoptosis-inducing effect, which may be mediated through death receptor pathway and mitochondrial pathway.