中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2015年
8期
1872-1875
,共4页
张玲莉%邱振鹏%涂毅%彭燕
張玲莉%邱振鵬%塗毅%彭燕
장령리%구진붕%도의%팽연
羽扇豆醇%HepG2细胞株%Wnt信号通路%抗增殖
羽扇豆醇%HepG2細胞株%Wnt信號通路%抗增殖
우선두순%HepG2세포주%Wnt신호통로%항증식
Lupeol%HepG2%Wnt signaling pathway%Antiproliferation
目的 探讨羽扇豆醇Lupeol通过Wnt/β-连环蛋白(β-catenin)信号通路抑制肝癌细胞增殖的作用机制.方法 采用细胞计数试剂盒(CCK-8)法及碘化丙锭(PI)单染法检测Lupeol对肝癌细胞的生长抑制作用及细胞周期的影响;以荧光素酶TOPflash/FOPflash报告系统检测Lupeol对HepG2细胞中Wnt信号通路的影响;Western blot法检测Lupeol对HepG2细胞中β-catenin蛋白表达的影响;荧光定量聚合酶链反应(Real-time PCR)检测Lupeol处理后c-Myc及细胞周期素D1(Cyclin D1)的mRNA表达水平;运用pGL3-Basic荧光素酶报告系统研究Lupeol对Cyclin D1启动子活性的影响.结果 Lupeol作用48h后,对HepG2细胞具有显著的抑制作用[细胞半数致死量半数抑制剂量(IC50)为(57.2±5.0) μmol/L].Lupeol处理HepG2细胞24、48 h使S期的细胞分布百分比上升至27％及42％;Top/Fop flash荧光素酶实验显示Lupeol使HepG2细胞中T细胞因子/淋巴增强因子(TCF/LEF)经典Wnt信号通路关键蛋白β-catenin介导的转录活性降低80％,并使细胞内累积的β-catenin的蛋白表达下调约30％,并减少了c-Myc及Cyclin D1的mRNA表达;同时其作用于Cyclin D1启动子使Cyclin D1的转录激活降低了3倍.结论 Lupeol可作用于Wnt/β-catenin信号通路进而抑制HepG2细胞增殖并呈现S期阻滞效应,其机制可能与减少β-catenin在细胞内累积及降低c-Myc与Cyclin D1的表达有关.
目的 探討羽扇豆醇Lupeol通過Wnt/β-連環蛋白(β-catenin)信號通路抑製肝癌細胞增殖的作用機製.方法 採用細胞計數試劑盒(CCK-8)法及碘化丙錠(PI)單染法檢測Lupeol對肝癌細胞的生長抑製作用及細胞週期的影響;以熒光素酶TOPflash/FOPflash報告繫統檢測Lupeol對HepG2細胞中Wnt信號通路的影響;Western blot法檢測Lupeol對HepG2細胞中β-catenin蛋白錶達的影響;熒光定量聚閤酶鏈反應(Real-time PCR)檢測Lupeol處理後c-Myc及細胞週期素D1(Cyclin D1)的mRNA錶達水平;運用pGL3-Basic熒光素酶報告繫統研究Lupeol對Cyclin D1啟動子活性的影響.結果 Lupeol作用48h後,對HepG2細胞具有顯著的抑製作用[細胞半數緻死量半數抑製劑量(IC50)為(57.2±5.0) μmol/L].Lupeol處理HepG2細胞24、48 h使S期的細胞分佈百分比上升至27％及42％;Top/Fop flash熒光素酶實驗顯示Lupeol使HepG2細胞中T細胞因子/淋巴增彊因子(TCF/LEF)經典Wnt信號通路關鍵蛋白β-catenin介導的轉錄活性降低80％,併使細胞內纍積的β-catenin的蛋白錶達下調約30％,併減少瞭c-Myc及Cyclin D1的mRNA錶達;同時其作用于Cyclin D1啟動子使Cyclin D1的轉錄激活降低瞭3倍.結論 Lupeol可作用于Wnt/β-catenin信號通路進而抑製HepG2細胞增殖併呈現S期阻滯效應,其機製可能與減少β-catenin在細胞內纍積及降低c-Myc與Cyclin D1的錶達有關.
목적 탐토우선두순Lupeol통과Wnt/β-련배단백(β-catenin)신호통로억제간암세포증식적작용궤제.방법 채용세포계수시제합(CCK-8)법급전화병정(PI)단염법검측Lupeol대간암세포적생장억제작용급세포주기적영향;이형광소매TOPflash/FOPflash보고계통검측Lupeol대HepG2세포중Wnt신호통로적영향;Western blot법검측Lupeol대HepG2세포중β-catenin단백표체적영향;형광정량취합매련반응(Real-time PCR)검측Lupeol처리후c-Myc급세포주기소D1(Cyclin D1)적mRNA표체수평;운용pGL3-Basic형광소매보고계통연구Lupeol대Cyclin D1계동자활성적영향.결과 Lupeol작용48h후,대HepG2세포구유현저적억제작용[세포반수치사량반수억제제량(IC50)위(57.2±5.0) μmol/L].Lupeol처리HepG2세포24、48 h사S기적세포분포백분비상승지27％급42％;Top/Fop flash형광소매실험현시Lupeol사HepG2세포중T세포인자/림파증강인자(TCF/LEF)경전Wnt신호통로관건단백β-catenin개도적전록활성강저80％,병사세포내루적적β-catenin적단백표체하조약30％,병감소료c-Myc급Cyclin D1적mRNA표체;동시기작용우Cyclin D1계동자사Cyclin D1적전록격활강저료3배.결론 Lupeol가작용우Wnt/β-catenin신호통로진이억제HepG2세포증식병정현S기조체효응,기궤제가능여감소β-catenin재세포내루적급강저c-Myc여Cyclin D1적표체유관.
Objective To study the antiproliferative effects and the regulatingmechanism of Lupeol on hepatocellular carcinoma cells in vitro.Methods Three kinds of liver cancer cells were treated with different concentration from 10-100 μmol/L and cell counting kit-8 (CCK-8) assay was employed to evaluate the antiproliferative effects.Propidium iodide (PI) staining assay was perferm to observe the mitotic cycle in Lupeol-treated HepG2 cells.T-cell factor/lymphocyte enhancer factor (TCF/LEF) transcriptional activity was assessed by TOPflash/FOPflash luciferase reporter plasmid kit.The β-catenin protein expression,mRNA expression of c-Myc and Cyclin D1 were determined by using Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR).pGL3-Basic vector loading with Cyclin D1 promoter region was transfected into HepG2 cells to clarity the effects of Lupeol on Cyclin D1 transcriptional activity.Results Lupeol dose-dependently suppressed the proliferation HepG2 cells and the 50％ inhibitory dose (IC50) is (57.2 ± 5.0) μmol/L.The cell percentage of S phase was significantly increased to 42％ after 48 h treatment of Lupeol.Top/Fop flash luciferase assay showed that Lupeol could supress the strongly activating status in HepG2.The β-catenin protein expression was decreased to approximately 70％ accompanied with the down-regulation of c-Myc and Cyclin D1 mRNA expression.The transcriptional activity of Cyclin D1 was also decreased to 25％ when cells were administrated with Lupeol,compared with control group.Conclusion Lupeol could inhibit the HepG2 cell proliferation and regulate the cell cycle in vitro via suppressing the Wnt/β-catenin signaling pathway,which was involved in suppressing expression of β-catenin,c-Myc and Cyclin D1.
