【摘要】 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抑制率的下降快更多还原

【Abstract】 Objective To investigate whether specific deoxyribozymes aimming at hepatitis C virus(HCV)5′-noncoding region(5′-NCR)have efficient inhibition effect on the target RNA in transgene HepG2.9706 cells. Methods 5′-GGCTAGCTACAACGA-3′ motif was adopted as the deoxyribozymes’ active center,and four sequences containing the characteristic sites 5′…R↓Y…3′(R=A/G,Y=U/C)were selected as the targets according to their location in the secondary structure of HCV 5′-NCR. Thus HCV-specific deoxyribozymes named DRz-232,DRz-127,DRz-84,DRz1 and their corresponding phosphorothioate ones(TDRz) and active-region-mutated phosphorothioate ones(MDRz)were designed and synthesizd. Lipofectin was used to introduce the drugs into HepG2.9706 cells containning plasmid pHCV-neo whose luciferase gene is under the control of HCV 5′-NCR and 5′-fragment of C region(5′-NCR-C). The cells were lysed at intended time points after the transfection of these drugs to release luciferase, and the reduction of luciferase activity were measured by chemiluminescence method for calculating inhibition rates of the drugs. DRzs/TDRzs with higher inhibition rates were selected for further study,including the time-effect relation-ships and comparison of results under different final concentrations of the transfected drugs. Results There was no significant difference between the effect of each DRz and its corresponding TDRz. DRz-127/TDRz-127 and DRz1/TDRz1 had higher inhibition rates than the other two DRzs/TDRzs,achieved 53.2%/ 50.6% and 44.7%/ 43.3% at the final concentration of 0.5 μmol/L for 24 hours. Inhibition rates of each couple of DRz/TDRz were higher than their corresponding MDRz,especially DRz-127/TDRz-127 to MDRz-127(P<0.05), and increased with the higher dose of drugs among the range from 0.25 to 0.75 μmol/L. The highest inhibition rates were achieved at 24 hours time point after transfection, but decreased notably after three days,especially that of DRz,other than TDRz. Conclusions Specific deoxyribozymes with suitable target sites may have both antisense inhibition and cleavage effect in transgene HepG2.9706 cells,thus become potentially a kind of perspective antiviral genetherapy tools. Appropriately phosphorothioated TDRzs almost have the same inhibition effect of and may be more stable than their corresponding DRzs.更多还原