硫代反义寡核苷酸体外对HDV的抑制作用

Inhibitory effect of HDV by antisense phosphorothioate oligodeoxynucleotide in vitro

目的观察硫代反义寡核苷酸 (S- ASODN)体外对 HDV的抑制作用。方法在 HDV/ HBV感染人胎肝细胞中加入不同浓度的针对 HDV Stem 区 6 84～ 6 98位核苷酸的 15聚 S- ASODN,分别采用 EL ISA和斑点杂交法检测上清液中 HDAg和细胞中 HDV RNA。结果 HBs Ag、HDAg在感染后第 2 d至第 16 d均可测出 ,以第 4d至第 12 d达高峰。加入 S- ASODN(2、4、6 μmol/ L)后 2 d,肝细胞中 HDV复制受到抑制 ,培养上清中 HDAg分泌量也减少 ,其抑制率呈剂量依赖关系 ;在同等剂量时(4μmol/ L) ,S- ASODN作用 2 d、4d、6 d,对 HDV抑制率大至相同。结论 HDV在原代培养人胎肝细胞中能稳定复制和表达至少达 12 d;S- ASODN能有效抑制 HDV/ HBV感染人胎肝细胞中 HDV复制与表达。本研究为进一步在动物体内研究 S- ASODN抗HDV作用和向临床过渡提供了有益的实验依据。

ObjectiveTo study inhibitory effect of replication and expression of HDV by antisense phosphorothioate oligodeoxynucleotide(S ASODN).MethodsA15 mer antisense phosphorothioate oligodeoxynucleotide(S ASODN)was complementary to stem I region(nucleotide 684～698) was added to edium of HDV/HBV infected human fetal hepatocytes.HDAg and HDV RNA were detected by ELISA,in situ hybridization and dot blot hybridization.Results HDAg,HBsAg could be detected from day 2 to day 16 after HDV/HBV infection.Secretion of HDAg reached summit from day 4 to day 12 after infection.Day 2 after S ASODN(2、4、6μmol/L)was added into the medium of hepatocytes,HDV RNA was inhibited.Secretion of HDAg in the supernant was reduced.Inhibitory rate showed the feature of dosage dependence.ConclusionPrimary human fetal hepatocyte is competent for infection with HDV/HBV,HDV can stably replicate and express in human fetal hepatocytes,and at least this replication and expression can reach for 12 days S ASODN can availably inhibit replication and expression of HDV in HDV/HBV infected human fetal hepatocytes.This study may be valuble for further research in animal and in clinic use. [