论文部分内容阅读
目的:探讨针对丙型肝炎病毒(hepatitis C virus,HCV)C基因的锁核酸核酶对病毒RNA复制与表达的特异性抑制作用.方法:设计合成能切割HCV C基因位点的DNAzyme、硫代DNAzyme和LNAzyme.实验设对照组和实验组.对照组包括空白对照组、脂质体对照组和脂质体-无关LNAzyme对照组.实验组包括脂质体-DNAzyme、脂质体-硫代DNAzyme组和脂质体-LNAzyme组.以阳离子脂质体介导转染hepG2.9706细胞.采用荧光定量PCR和化学发光技术分别监测24、48、96 h细胞培养上清液中HCV RNA含量及荧光素酶基因表达;四甲基偶氮唑蓝(MTT)法监测细胞代谢.结果:加入药物后,脱氧核酶、硫代脱氧核酶及锁核酸核酶组对HCV RNA复制和荧光素酶基因表达的抑制作用均较对照组强(P<0.01),其中,锁核酸核酶的抑制作用均较脱氧核酶及硫代脱氧核酶明显(P<0.05),平均抑制率分别达47.55%和52.44%,且随用药时间延长,抑制率呈增高趋势,96 h后,HCV RNA复制和荧光蛋白表达的下降率均较用药前明显(P<0.01),其中,锁核酸核酶的抑制作用均较脱氧核酶及硫代脱氧核酶明显(P<0.05),平均下降率分别达79.40%和80.05%.而LNAzyme对细胞活性基本无影响.结论:LNAzyme能特异性抑制HCV C基因的复制与表达,且优于硫代修饰的DNAzyme.
OBJECTIVE: To investigate the specific inhibitory effect of the locked nucleic acid ribozyme against C gene of hepatitis C virus (HCV) on the replication and expression of viral RNA.Methods: DNAzyme designed to cleave the HCV C gene was designed and synthesized, DNAzyme and LNAzyme.Experimental control group and experimental group.The control group included blank control group, liposome control group and liposome-unrelated LNAzyme control group.Experimental group included liposome-DNAzyme, liposome-thio DNAzyme Group and lipofectamine-LNAzyme group.The cationic liposomes were transfected into hepG2.9706 cells.The fluorescence quantitative PCR and chemiluminescence were used to detect the content of HCV RNA and fluorescence in 24, 48 and 96 h cell culture supernatants respectively (MTT) method was used to monitor the cell metabolism.Results: After addition of drugs, the activity of HCV RNA replication and luciferase gene (P <0.01). The inhibitory effect of the locked nucleic acid ribozyme was significantly (P <0.05) higher than that of the DNAzyme and the sulfur deoxyribozyme, with the average inhibition rates of 47.55% and 52.44%. With the prolongation of treatment time, the inhibition rate showed an increasing trend, 96 h (P <0.01). The inhibitory effect of the locked nucleic acid ribozyme was more obvious than that of the DNAzyme and the sulfur deoxyribozyme (P <0.05) The average rate of decrease was 79.40% and 80.05%, respectively, while LNAzyme had no effect on cell activity.Conclusion: LNAzyme can specifically inhibit the replication and expression of HCV C gene, and is superior to the thio-modified DNAzyme.