摘要
目的:利用RNAi技术特异性的抑制NF-κB亚单位p65的表达,观察其对p65表达的抑制作用及联合5-FU对食管鳞癌细胞Eca109和EC9706的影响.方法:将终浓度为50nmol/L的p65 siRNA转染到食管鳞癌细胞EC9706和Eca109中,通过RT-PCR检测0、24、48和72h时段p65 mRNA的表达水平.Western blotting法检测p65和Bcl-2蛋白表达,AnnexinV/PI复染结合流式细胞仪检测细胞凋亡,显微镜下观察p65siRNA与5-FU单独或联合应用对食管鳞癌细胞形态学特性的影响.结果:EC9706和Eca109细胞转染p65 siRNA24、48和72h后,p65 mRNA的表达水平随时间的延长逐渐下调,在72h的阻断效率最为明显,与0h相比,差异有显著性(0.12±0.01vs0.28±0.05,0.1±0.01vs0.38±0.04,均P<0.05),转染72h后,p65和Bcl-2蛋白表达水平下调.EC9706和Eca109转染p65 siRNA后,细胞凋亡指数明显升高(6.65%±0.27%vs2.03%±0.08%,8.03%±0.06%vs2.66%±0.25%,均P<0.05);p65siRNA转染72h后,EC9706和Eca109细胞增殖较慢;当p65 siRNA与5-FU联合作用,细胞增殖明显受到抑制.结论:p65 siRNA可阻断NF-κB信号通路,下调NF-κB下游基因中抗凋亡蛋白Bcl-2的表达,表明活化的NF-κB信号通路可成为食管鳞癌基因治疗中一个重要的分子靶点.
AIM：To evaluate the inhibitory effects of nuclear factor-kappa B p65（NF-κB p65）on expression of p65 and to determine its combinatory effects with 5-FU on proliferation and apoptosis in EC9706 and Eca109 esophageal squamous cell carcinoma（ESCC）.
METHODS：EC9706 and Eca109 cells were transfected with 50 nmol/L p65 siRNA.The expression level of p65 mRNA was measured using PT-PCR at 0,24,48 and 72 h.P65 and Bcl-2 protein levels were determined using Western blotting. The cells were also stained with FITC-annexin V and PI,and cancer cell apoptosis was detected by flow cytometry using CellQuest acquisition and analysis programs.Morphological changes of ESCC cells were observed microscopically following p65 siRNA transfection with or without 5-FU.
RESULTS：For EC9706 and Eca109 transfected with p65 siRNA,the expression level of the p65 mRNA in ESCC cells was down-regulated with time.Peak inhibitory effect occured at 72 h and a significant difference was detected,compared with 0 h（0.12±0.01 vs 0.28±0.05,0.1±0.01 vs 0.38±0.04,both P〈0.05）.The protein levels of p65 and Bcl-2 decreased after transfection with p65 siRNA at 72 h.There was a significant increase in apoptosis level at 72 h following p65 siRNA transfection（6.65%±0.27%vs 2.03%± 0.08%,8.03%±0.06%vs 2.66%±0.25%,both P 〈0.05）.The proliferation of EC9706 and Eca109 was slow after transfection with p65 siRNA at 72 h,while p65 siRNA in combination with 5-FU significantly inhibited cell proliferation.
CONCLUSION：p65 siRNA can block NF-κB signaling pathway,down-regulates expression of Bcl-2,which makes activated NF-κB pathway a potential key target in gene therapy for ESCC.
出处
《世界华人消化杂志》
CAS
北大核心
2008年第16期1716-1721,共6页
World Chinese Journal of Digestology
基金
教育部"十五"211工程重点建设资助项目
No.2002-20~~
