反义寡核苷酸体外抑制丙型肝炎病毒的研究

Inhibition of hepatitis C virus by antisense oligodeoxynucleotide in vitro

研究反义寡核苷酸体外抑制丙型肝炎病毒（ＨＣＶ）的方法。方法以重组丙型肝炎病毒（ｐＣＤ－ＨＣＶ）转染的Ｈ９细胞为对象，通过半定量逆转录聚合酶链反应和ｄｏｔＥＬＩＳＡ检测ＨＣＶｍＲＮＡ和抗原表达的变化，观察与ＨＣＶ结构区（包括ＡＵＧ）互补和同源以及随机的硫代磷酸化寡核苷酸（ＰＳ－ＡＳＯＮ、ＰＳ－ＯＤＮ、ｒＰＳ－ＯＤＮ）对ＨＣＶ的抑制作用。结果ＰＳ－ＡＳＯＮ和ＰＳ－ＯＤＮ可有效地进入靶细胞并在体外与靶基因杂交结合，终浓度为１０μｍｏｌ／Ｌ的ＰＳ－ＯＤＮ、ｒＰＳ－ＯＤＮ对ＨＣＶ均无抑制作用，但ＰＳ－ＡＳＯＮ可明显降低ＨＣＶｍＲＮＡ和抗原表达水平，具有药物浓度和时间依赖效应，脂质体修饰可增强ＰＳ－ＡＳＯＮ的反义抑制作用，磷酸钙修饰却无此作用。结论与ＨＣＶ互补的ＰＳ－ＡＳＯＮ是ＨＣＶ的反义寡核苷酸，在翻译水平上具有反义抑制作用。

Objective To study inhibitory effect of antisense oligodeoxynucleotide (asODN) on HCV in vitro. Methods The H9 cells transfected by pCD HCV, a recombinant HCV containing total HCV structural gene, were treated with two 15 mers phosphorothioate (PS) ODNs complementary (PS ASON) and homologous (PS ODN) to HCV core genomic region, which were labeled with digoxin (DIG). Spot blot hybridization was carried out. Treated by the two ODNs, rPS ODN (a 15 mers PS ODN of random sequence) or PS ASON were modified with two liposomes (DOTAP and Lipofectin) and calcium phosphate precipitation respectively. With a half ration, the variation of level of HCV mRNA and HCV antigen expression was observed by RT PCR and dot ELISA. 3H TdR adding test was done to observe PS ASON cytotoxicity. Results PS ODN and PS ASON were detected in the H9 cells. The target gene was hybridized to PS ASON and PS ODN labeled with DIG. PS ASON cut down level of HCV mRNA and HCV antigen expression obviously. However, PS ODN and rPS ODN did not influence the level of the both. The time dependent and dose dependent inhibition of PS ASON was observed. In contrast to free PS ASON, both of liposomal PS ASON showed more highly effective inhibition, but calcium phosphate precipitation PS ASON complex did not. The results showed PS ASON did not influence the H9 cells growth at 10 μmol/L. Conclusion PS ASON complementary to HCV core gene is asODN and exerts antisense inhibitory effect on the level of HCV translation obviously, but not on the level of HCV replication and transcription.