短发夹RNA抑制肝癌细胞HepG_2 COX-2基因表达的探讨  

The Studies on Depressant Effect of Short-Hairpin RNA of COX-2 Gene Expression in HepG2 Cells

作　　者：杨义明[1] 刘预[2] 涂植光[1] 陈亚芹[1] 刘靳波[1] 

机构地区：[1]重庆医科大学检验系临床检验诊断学省部共建教育部重点实验室,重庆市400016 [2]重庆市肿瘤研究所临床检验中心

出　　处：《中国肿瘤临床》2007年第10期566-569,共4页Chinese Journal of Clinical Oncology

基　　金：重庆市科委自然科学基金资助(编号:2006BB5271)

摘　　要：目的:研究shRNA对肝癌细胞株HepG2中COX-2基因表达的抑制作用,同时检测对细胞周期和细胞生长的影响。方法:设计、合成一对环氧化酶-2(COX-2)基因编码的反向重复序列,中间间隔9个核苷酸序列,定向克隆至质粒载体pGenesil-1,构建抑制COX-2基因的短发夹RNA(short hairpin RNA shRNA)的重组表达质粒pshRNA-COX-2,用脂质体LipofectamineTM2000转染肝癌细胞HepG2,经RT-PCR和Westernblot分别检测pshRNA-COX-2重组表达质粒对COX-2mRNA和蛋白抑制效应,流式细胞仪检测细胞周期、细胞计数检测细胞生长变化。结果:pshRNA-COX-2重组表达质粒能够抑制COX-2基因表达。与未转染肝癌细胞HepG2相比,pshRNA-COX-2重组表达质粒转染肝癌细胞后,COX-2mRNA和蛋白表达明显降低,抑制率分别为69.9%和50.3%;G0-G1期细胞由55.4%上升为77.9%,S期细胞由31.1%下降为16.2%;细胞生长明显减慢。结论:构建的pshRNA-COX-2重组表达载体能够显著抑制COX-2基因表达;引起G0-G1期细胞增多,S期细胞减少,从而阻止肝癌细胞生长。Objective： To investigate the inhibitory action of short hairpin RNA （ShRNA） targeting at the COX-2 gene expression in HepG2 cells and to determine the effect of ShRNA on the change of cell cycle and cell growth. Methods： A pair of reverse repeated sequence targeting COX-2 mRNA were contrived and synthesized, with 9 nucleotide sequences between them, and were directionally cloned to the pGenesil-1, a plasmid vector to construct a recombinant expression plasmid pshRNA- COX-2, and LipofectamineTM2000, a liposome was used to transfect the hepatocelluar carcinoma cell HepG2. Suppressing effects of the pshRNA-COX-2 on the COX-2 mRNA and proteins were detected by the RT-PCR and Western blot methods, respectively. Flow cytometry was conducted to assay the cell cycle and change of the cell growth. Results： Recombinant expression plasmid pshRNA-COX-2 could inhibit the expression of COX-2 gene. Compared to the group without transfecting the hepatoma carcinoma cell （HCC） HepG2, the expressions of COX-2 mRNA and proteins were apparently lower in the group with recombinant expression plasmid pshRNA-COX-2＇s transfecting the HCC. The inhibition rates were 69.9% and 50.3% respectively. The cell counts of the GO and G1 phase were increased from 5.4% to 77.9%, and that of stage S was decreased from 31.1%to16.2 %. The cell growth became significantly slow. Conclusion： Construction of the recombinant expression vector pshRNA-COX-2 could significantly inhibit the expression of COX-2 gene, which result in an increase of the cells in the GO and G1 phase and a decrease of the cells in the S phase, thus suppressing the proliferation of hepatocelluar cells.

关 键 词：短发夹RNA 环氧化酶-2 重组表达质粒 细胞周期 

分 类 号：R735.7[医药卫生—肿瘤]