柠檬酚对人肝癌HepG_2细胞增殖的影响

Effect of Citrusinol on Proliferation of Human Hepatocellular Carcinoma Cells HepG_2

为了探讨柠檬酚对人肝癌HepG_2细胞增殖的影响,本文利用四甲基偶氮唑蓝(MTT)法、细胞生长曲线以及细胞克隆形成实验检测了柠檬酚对细胞生长和克隆形成的影响,并应用HE、吖啶橙染色、流式细胞术以及激光扫描共聚焦显微镜分别观察和检测了不同浓度柠檬酚作用后细胞形态、细胞周期和细胞骨架蛋白F-actin的变化。结果显示,柠檬酚能明显抑制人HepG_2细胞生长,且呈浓度依赖性,其IC50值为79.14μmol/L,柠檬酚给药组细胞克隆率明显明显低于空白对照组;不同浓度的柠檬酚在作用HepG_2细胞24h后,细胞均出现了明显的皱缩、胞浆空泡化和细胞核固缩的形态学改变;同时,70μmol/L和80μmol/L的柠檬酚处理组G2/M期细胞比例显著高于对照组(22.72±1.07,28.68±1.36 vs 16.34±0.66,P<0.01);而共聚焦显微镜观察发现,柠檬酚能引起HepG_2细胞骨架蛋白F-actin的解聚和断裂。综上,推测柠檬酚可能是通过破坏细胞的Actin骨架,阻止细胞有丝分裂,细胞周期阻滞在G2/M期,从而抑制人肝癌HepG_2细胞的增殖。

In order to study the effect of citrusinol on human hepatocellular carcinoma cells HepG2,terazolium salt colorimetry assay（MTT ）,cell growth curve and colony formation assay was used to detected the proliferation and colony formation.The morphology of cells was observed by HE staining and Acridine orange staining.Cell cycle changes were detected by Flow Cytometry（FCM）.The morphological changes of cytoskeletal protein F-actin were observed by laser confocal microscopy.The results showed thatcitrusinol inhibited HepG2cells proliferation and colony formation in a dose-dependent manner.IC50 of citrusinol was 79.14 μmol/L after 24 hours.The morphology of HepG2 cells was changed,showing cell shrinkage,cytoplasm vacuoles and nuclear chromatin pyknosis.The percentage of G2/M phase of HepG2 cells treated with 70,80 μmol/L citrusinol was significantly higher than that of the control group （22.72±1.07,28.68±1.36 vs 16.34±0.66,P〈0.01）.F-actin cytoskeletal protein was showed from polymerization to depolymerization after citrusinol treatment.Based on the experimental results,it was concluded that citrusinol can prevent cell mitosis to arrest the cell cycle in G2/M phase,by disrupting Actin cytoskeleton,which may be one of the mechanisms of citrusinol＇s anti-tumor effect.