反义寡核苷酸靶向肝细胞体外抗乙型肝炎病毒的实验研究  

Inhibtion of hepatitis B viral gene expression and repli-cation in vitro by targeted antisense Oligonucleotides

作　　者：钟森[1] 张定凤[1] 温守明[1] 王升启[1] 王玉芝[1] 陈学华[1] 黄爱龙[1] 任红[1] 周晓东[1] 

机构地区：[1]重庆医科大学病毒性肝炎研究所,沪洲医学院附属医院,解放军空军总医院

出　　处：《中华医学杂志》1995年第7期392-395,共4页National Medical Journal of China

基　　金：国家自然科学基金;中国人民解放军总后勤部卫生部"八五"招标课题基金

摘　　要：作者在对２．２．１５细胞中乙型肝炎病毒（ＨＢＶ）基因测序的基础上，设计合成了１段针对Ｕ５样序列区的１６－聚硫代磷酸反义寡核苷酸（ＡＳＯＮ），并将其与肝导向性载体——半乳糖化白蛋白和半乳糖化多聚赖氨酸连接，在２．２．１５细胞中观察了它们的抗病毒作用。在相同实验条件下，非导向组ＡＳＯＮ对表面抗原和ｅ抗原的抑制仅为７０％和５８％，而导向组ＡＳＯＮ可高达９０％～９６％和７９％～８２％，两者差异有显著意义。可见肝导向性载体能明显提高ＡＳＯＮ的抗病毒活性。ＡＳＯＮ对ＨＢＶ的抑制作用是特异性的，呈剂量依赖性关系；而对肝细胞本身的甲胎蛋白合成无影响，亦未见细胞毒性作用。无关序列寡核苷酸无明显抑制作用，反义抑制机理可能是作用在病毒逆转录和翻译水平上。e chose the 2. 2. 15 cells as an in vitro cell cultureassay system, and identified the surface antigen subtypeof hepatitis B virus (HBV) DNA in the cells, by usingthe amplification of polymerase chain reaction and di-rected sequencing of amplified products. According tothe result of the sequencing , a 16-mer phosphorothioateanalogue of the antisense oligonucleotide (PS-ASON)directed against the HBV US-unique region was synthesized and then linked with two liver-targting ligands.The galactosylated human serum albumin coupled poly-L-lysine and the galactosylated polyLlysine. The ef-fect of different modifications of ASONs on the expres-sion of HBV gene was compared by using the 2. 2. 15cells. In the same experimental conditions, the inhibitary effects of surface antigen (HBsAg) and antigen(HBeAg) by PSASON were 70%0 and 58%0 respective-ly, and those of HBsAg and HBeAg by ligand-PS-A-SONs were 90%～ 96%0 and 78%～ 82%. In the sametime, the amount of HBV NDA in culture supernatantand cells was depressed significantly- Thus, the ligandstargted ASON to the hepatocytes were more effectiveinhibitors of HBV gene expression- ASONs were effec-tive and specific inhibitors of HBV replication and expression, caused the dose-dependent inhibition of viralproteins and had no effect on cellular α-fetoprotein syn-thesis and no cytotoxicity.

关 键 词：乙型肝炎病毒 反义寡核苷酸 基因表达调控 

分 类 号：R512.620.3[医药卫生—内科学]