摘要目的 观察氧化苦参碱对HBV DNA聚合酶活性的影响,探讨其抗HBV的可能机制.方法 从HepG2.2.15细胞培养上清液中获取HBV颗粒,以包括32P-dCTP在内的脱氧核酸作为底物,加入不同药物浓度的反应液,氧化苦参碱的浓度分别为1000 μg/ml、800 μg/ml、600 μg/ml、400 μg/ml和200 μg/ml,阿德福韦酯的浓度分别为100 μg/ml、80 μg/ml、60 μg/ml、40 μg/ml和20 μg/ml,并设立空白对照组,每组3个复管.于37℃孵育过夜,蛋白酶K消化后每管取35 μl反应液点样于DE 81滤膜上,用液体闪烁测量仪测量掺入HBV DNA正链中的32P-dCTP的放射量.结果 与空白对照组相比,不同浓度的氧化苦参碱作用后,HBV DNA聚合酶活性维持在103%～107%;不同浓度的阿德福韦酯作用后,HBV DNA聚合酶活性维持在91%～102%之间.各组之间比较差异无统计学意义(P>0.05).结论 氧化苦参碱在体外对HBV DNA聚合酶活性无直接抑制作用.

abstractsObjective To determine the effect of oxymatrine on the activity of HBV DNA polymerase in vitro. Methods Hepatitis B virus particles were purified from supernatant of cultured HepG2.2.15 cells by uhracentrifugation, and then were mixed with reaction buffer containing NP-40, β-mercaptoethanol, 32P-labelled nucleoside triphosphate (dCTP), MgCl2, and different concentrations of oxymatrine ( 1000 μg/ml, 800 μg/ml, 600 μg/ml, 400 μg/ml and 200 μg/ml) or adefovir dipivoxil ( 100 μg/ml, 80 μg/ml and 60 μg/ml, 40 μg/ml and 20 μg/ml). After incubation at 37 ℃ overnight, proteinase K was added to the reaction system for digestion and 35 μl of samples were spotted onto DE81 paper. Activities of endogenous polymerase in HBV particles were assessed by determining the radioactivity of 32P-labelled dCTP incorporated in the plus-strain of viral DNA. Results Compared with the blank control, the activity of endogenous polymerase in HBV particles treated with different doses of oxymatrine varied from 103% to 107%, and it varied from 91% to 101% when treated with different doses of adefovir dipivoxil. No significant difference was observed among treated groups and the control (P > 0. 05 ). Conclusion No direct inhibitory effect of oxymatrine on the activity of HBV polymerase was observed in vitro.