摘要
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.
出处
《中华医学杂志》
CAS
CSCD
北大核心
1997年第8期567-570,共4页
National Medical Journal of China
基金
国家自然科学基金
国家教委归国留学人员科研启动基金
关键词
寡核苷酸类
反义
丙型肝炎病毒
体外抑制
Hepatitis C viruses Oligo nucleotide, antisense Antisense inhibitory effect