CAJ | 학술논문

目的建立肝癌顺铂耐药细胞系,探讨Wnt/β-catenin信号通路在肝癌顺铂耐药中的作用.方法使用顺铂逐步增加剂量法诱导人肝癌HepG-2细胞,以建立其多药耐药细胞株HepG-2/DDP；MTT细胞毒实验检测顺铂对HepG-2和HepG-2/DDP细胞的生长抑制作用；荧光定量-PCR检测β-catenin 基因的表达；基因转染双链SiRNA干扰β-catenin基因的表达；Western blot实验检测目的蛋白的表达改变.结果成功建立了人肝癌HepG-2顺铂耐药细胞株；MTT细胞毒实验结果显示顺铂对HepG-2和HepG-2/DDP细胞的IC50值分别为(2.29±0.14) μmol/L和(20.51±0.84)μmol/L(t=95.68,P＜0.01),与HepG-2细胞比较,HepG-2/DDP细胞对顺铂耐药8.96倍.荧光定量PCR结果显示:HepG-2和HepG-2/DDP细胞中β-catenin基因表达的2-△Ct值分别为0.323±0.065和0.674±0.097(P＜0.01),Western blot检测也显示β-catenin蛋白的表达在HepG-2/DDP细胞也显著高于HepG-2细胞.化学合成的靶向SiRNA可以显著下调β-catenin的表达,顺铂对照组、SiRNA靶向干扰组、SiRNA阴性干扰组HepG-2/DDP细胞的IC50值分别为(21.02±1.64)、(6.23±0.68)、(20.44±1.26) μmol/L,对照组和SiRNA靶向干扰组之间差异有统计学意义(P＜0.01).结论顺铂耐药细胞HepG-2/DDP出现了Wnt/β-catenin信号通路的激活,SiRNA干扰β-catenin基因表达可显著提高顺铂耐药细胞HepG-2/DDP对顺铂的敏感性,为克服肝癌顺铂新靶点的寻找提供了理论依据.
목적 건립간암순박내약세포계,탐토Wnt/β-catenin신호통로재간암순박내약중적작용.방법 사용순박축보증가제량법유도인간암HepG-2세포,이건립기다약내약세포주HepG-2/DDP；MTT세포독실험검측순박대HepG-2화HepG-2/DDP세포적생장억제작용；형광정량-PCR검측β-catenin 기인적표체；기인전염쌍련SiRNA간우β-catenin기인적표체；Western blot실험검측목적단백적표체개변.결과 성공건립료인간암HepG-2순박내약세포주；MTT세포독실험결과현시순박대HepG-2화HepG-2/DDP세포적IC50치분별위(2.29±0.14) μmol/L화(20.51±0.84)μmol/L(t=95.68,P＜0.01),여HepG-2세포비교,HepG-2/DDP세포대순박내약8.96배.형광정량PCR결과현시:HepG-2화HepG-2/DDP세포중β-catenin기인표체적2-△Ct치분별위0.323±0.065화0.674±0.097(P＜0.01),Western blot검측야현시β-catenin단백적표체재HepG-2/DDP세포야현저고우HepG-2세포.화학합성적파향SiRNA가이현저하조β-catenin적표체,순박대조조、SiRNA파향간우조、SiRNA음성간우조HepG-2/DDP세포적IC50치분별위(21.02±1.64)、(6.23±0.68)、(20.44±1.26) μmol/L,대조조화SiRNA파향간우조지간차이유통계학의의(P＜0.01).결론 순박내약세포HepG-2/DDP출현료Wnt/β-catenin신호통로적격활,SiRNA간우β-catenin기인표체가현저제고순박내약세포HepG-2/DDP대순박적민감성,위극복간암순박신파점적심조제공료이론의거.
Objective To establish a cisplatin (DDP)-resistant HepG-2 cell line,and to explore the role of Wnt/β-catenin signaling pathway on multidrug resistance in human hepatocellular carcinoma.Methods The HepG-2 cells were exposed in a gradually increasing dose of DDP to establish a cisplatin ( DDP)-resistant HepG-2 cell line.MTT assay was used to detect the cytotoxic activity of DDP against HepG-2 and HepG-2/DDP cells.The mRNA expression of β-catenin was determined by Real-time PCR assay.The small interfering RNA was used to specifically knockdown β-catenin expression in HepG-2/DDP cells.The protein expression was detected by western blot analysis.Results The DDP-resistant cell line HepG-2/DDP was established by gradient DDP induction successfully.The IC50 values of DDP against HepG-2 and HepG-2/DDP cells were (2.29 ± 0.14) μmol/L and ( 20.51 ± 0.84 ) μmol/L,respectively ( t=95.68,P＜0.01 ),HepG-2/DDP cells was 8.96 times than HepG-2 cells on the resistance of cisplatin.The result of real time PCR showed that 2-△Ct value of β-catenin in HepG-2 cells and HepG-2/DDP cells were (0.323±0.065) and (0.674 ±0.097) (P＜0.01 ).And the protein expression of cisplatin in HepG-2/DDP cells was also significantly higher than that in the HepG-2 cells.The expresssion of β-catenin was significantly and specifically depleted by siRNA duplexes(P＜0.01 ).The IC50 values of cisplatin against HepG-2/DDP cells were (21.02 ± 1.64) μmol/L in cisplatin control group,(6.23 ± 0.68 ) μmol/L in SiRNA targeting interference group and ( 20.44 ± 1.26 ) μmol/L in SiRNA negative interference group,and there was significant difference between control group and SiRNA targeting interference group ( P＜0.01 ).Conclusion The Wnt/β-catenin signaling pathway was activated on the cisplatin(DDP)-resistant HepG-2 cell line and down regulation of β-catenin increased the chemosensitivity of HepG-2/DDP cells against cisplatin.It provided a theoretical basis for finding the new targets of multidrug resistance in liver cancer.