【摘要】 目的 探讨丁型肝炎病毒(HDV)核酶在细胞内抑制丙型肝炎病毒(HCV)RNA的可能性。方法 针对HCV-5′NCR-C RNA的不同靶位构建了pC1-RzC1(107～113nt)、pC1-RzC2(268～274nt)、pC1-RzC3(345～351nt)3个HDV核酶重组真核表达载体,采用脂质体介导将它们转染至HCV阳性胎肝细胞中,通过荧光定量聚合酶链反应(PCR)检测细胞和培养上清液中的HCV RNA,初步探讨HDV核酶对HCV RNA的抑制活性。结果 (1)重组表达载体通过PCR、酶切和碱基序列测定证实了3组HDV核酶定向插入到真核表达载体。(2)免疫组织化学观察到HCV阳性胎肝细胞中HCV NS3、NS5抗原及逆转录-PCR法检测胎肝细胞及上清液中的HCV RNA正、负链,同时利用Light Cycler荧光PCR定量检测HCV RNA的含量,证明HCV感染胎肝细胞成功。(3)HCV阳性胎肝细胞中HDV核酶对全长HCV RNA的抑制活性,当剂量为0.5μmol/L时,pC1-RzC1、pC1-RzC2、pC1-RzC3抑制活性分别为53.2%、50.5%、10.6%(t=5.25,P<0.01)。pC1-RzC1连续1周作用于HCV阳性的胎肝细胞,结果显示第2～7天的抑制活性分别是60.7%、64.2%、68.4%、71.9%、78.8%、83.1%(t=15.34,P<0.01)。结论 pC1-RzC1、pC1-RzC2在HCV阳性胎肝细胞中对HCV RNA的抑制活性明显高于pC1-RzC3。更多还原

【Abstract】 Objective To investigate whether HDV ribozymes can intracellularly inhibit HCV RNA. Methods The mammalian expression vectors, pC1-RzC1, pC1-RzC2 and pC1-RzC3, containing ribozymes cDNA of RzC1, RzC2, and RzC3, were constructed targeting different HCV-5’ NCR-C RNA regions. Then the HCV-positive fetal hepatocytes were transfected with these plasmids using liposome-mediated method. The inhibitory effects of HDV ribozymes were evaluated by HCV RNA quantitation in cultured cells and the supernatants. Results (1)A11 the three HDV ribozymes were inserted into the expression vector. (2)Fetal hepatocytes were infected with HCV proven by RT-PCR and fluorescent quantitative PCR and expressed HCV NS3 and NS5 antigens by immunocytochemistry. (3)HDV ribozymes inhibited the activity of the target HCV RNA at expect positions in HCV-positive hepatocytes. At 0.5μmol/L , the inhibitory rate of pC1-RzC1, pC1-RzC2, and pC1-RzC3 was 53.2%, 50.5 %, and 10.6% respectively. pC1-RzC1 was used continuously for one week, showing the inhibitory rate of 60.7%, 64.2%, 68.4%, 71.9%, 78.8% and 83.1% on the 2nd, 3rd, 4th, 5th, 6th and 7th day. Conclusion The inhibitory activity of pC1-RzC1(107-113 nt) and pC1-RzC2 (268-274 nt) is greater than that of pC1-RzC3 (345-351 nt) in HCV-positive hepatocytes.更多还原