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目的 观察特异性脱氧核酶 (DRz)在丙型肝炎病毒 (HCV) 5′ 非编码区 (5′ NCR)转基因肝癌细胞株中对靶RNA的抑制活性。方法 采用 5′ GGCTAGCTACAACGA 3′基序为活性中心 ,分析HCV 5′ NCR中符合 5′…R↓Y… 3′(R =A/G ,Y =U/C)特征的位点及 5′ NCR的二级结构以确定靶位 ,设计并合成HCV靶向性脱氧核酶DRz 2 32、DRz 12 7、DRz 84、DRz1以及对应的两端被硫代修饰的DRz(TDRz)和活性中心区人工突变的硫代DRz(MDRz)。用脂质体转染法将导入HCV 5′ NCR及部分C区 (5′ NCR C)的转基因肝癌细胞株HepG2 .970 6 中 ,作用一定时间后 ,用化学发光法检测荧光素酶活性的变化 ,计算抑制率。选择抑制率较高者进行时效关系的观察和不同浓度抑制效果的比较。结果 各DRz和对应TDRz的活性无明显差别。DRz 12 7/TDRz 12 7和DRz1/TDRz1的抑制活性相对较高 ,终浓度为 0 .5 μmol/L作用 2 4h抑制率分别达 5 3.2 % / 5 0 .6 %和 4 4 .7% / 4 3.3%。各DRz/TDRz的抑制率均高于对应的MDRz ,其中DRz 12 7/TDRz 12 7与MDRz 12 7(4 1.2 % )相比抑制率有显著性差异 ,P <0 .0 5。在 0 .2 5～ 0 .75 μmol/L范围 ,抑制率随药物浓度的升高而增高。在所观察的时间点中 ,加药后 2 4h抑制率最高 ,3d后抑制率显著下降 ;DRz抑制率的下降快
Objective To observe the inhibitory activity of specific deoxyribozyme (DRz) on target RNA in a 5 ’noncoding (5’ NCR) transgenic hepatoma cell line of Hepatitis C virus (HCV). METHODS: The 5 ’GGCTAGCTACAACGA 3’ motif was used as the active site to analyze the 5 ’NCR of 5’ NCR and 5 ’NCR (TDRz) and active center of the active region were designed and synthesized by targeting the secondary structure of DRz 2 32, DRz 12 7, DRz 84, DRz 1 and corresponding DRz (TDRz) Mutant thio-DRz (MDRz). Transfection of HepG2.9706 cells transfected with HCV 5 ’NCR and partial C region (5’ NCR C) by lipofection method was followed by a change in luciferase activity by a chemiluminescence method after a certain period of action , Calculate the inhibition rate. Select the higher inhibition rate of the relationship between the observed and the effect of different concentrations of inhibition compared. Results There was no significant difference in the activity of each DRz and corresponding TDRz. The inhibitory rates of DRz 12 7 / TDRz 12 7 and DRz1 / TDRz1 were relatively high, with a final concentration of 0. 5 μmol / L for 24 h, respectively, reaching 5 3.2% / 56.6% and 44.7% / 50% 4 3.3%. The inhibition rate of each DRz / TDRz was higher than the corresponding MDRz, and the inhibition rate of DRz 12 7 / TDRz 12 7 was significantly different from that of MDRz 12 7 (4 1.2%), P <0. In the range of 0 .2 5 ~ 0. 75 μmol / L, the inhibition rate increased with the increase of the drug concentration. At the observed time points, the inhibition rate was the highest at 24 hours after dosing, and the inhibition rate was significantly decreased after 3 days. The decrease in DRz inhibition rate was faster