脱氧核酶及锁核酸核酶对2.2.15细胞中HBsAg和HBeAg表达的抑制作用

Inhibitory effects of DNAzyme and LNAzymeon expressi on of HBsAg and HBeAg in 2.2.15 cells

目的:观察针对HBV前C/C区的脱氧核酶(DNAzyme)及锁核酸核酶(LNAzyme)对2.2.15细胞中HBsAg、HBeAg表达的抑制作用。方法:设计合成针对HBV前C/C区2031位点的10-23DNAzyme、点硫代修饰的10-23DNAzyme及LNAzyme。本实验设对照组和10-23DNAzyme组、点硫代修饰的10-23DNAzyme组、LNAzyme实验组。对照组包括空白对照组、单纯脂质体对照组、单纯10-23DNAzyme对照组、无关10-23DNAzyme对照组。观察在0.16、0.64、1.28、1.60及1.92μmol.L-1浓度下及12、24、36、48、60、72、84及96 h时间段对2.2.15细胞的HBsAg、HBeAg表达的抑制效应。结果:10-23DNAzyme及LNAzyme作用于2.2.15细胞后,可明显抑制HBsAg、HBeAg的表达,且抑制率LNAzyme明显高于点硫代修饰的10-23DNAzyme,而后者又明显高于未进行任何修饰的10-23DNAzyme组。LNAzyme及点硫代修饰的10-23DNAzyme对HBsAg的最高抑制率分别是(91.6±8.4)%和(78.4±2.0)%,对HBeAg的最高抑制率分别是(90.1±5.2)%和(76.4±4.8)%;在给药后12 h就表现出抑制效应,48 h达高峰,LNAzymes、点硫代修饰的10-23DNAzyme有效抑制时间分别是为84及72 h。其对2.2.15细胞未见明显的细胞毒性作用。结论:10-23DNAzyme及LNAzyme对2.2.15细胞具有明显的高效阻断HBV的HBsAg、HBeAg表达的作用,且LNAzyme优于DNAzyme,是一类特异的高效的抗HBV治疗剂。

Objective To observe the suppression of HBsAg and HBeAg expression by DNAzyme and LNAzyme located at HBV pre-area of HBV. Methods Eecoding sequence of 10-23 DNAzyme thiolmodificated 10-23DNAzyme and LNAzyme that were directed against Pre C/C region of HBV were designed and synthesized, Experimental groups and control groups were set up. The experimental groups included 10-23 DNAzyme group, S-10-23 DNAzyme group and LNAzyme group. The control groups include blank control group, simple lipofectamine group, simple 10-23DNAzyme group and random 10-23 DNAzyme group. In the dosege of 0. 16, 0.64, 1.28, 1.60, 1.92 μmol·L^-1 and the time of 12, 24, 36, 48, 60, 72, 84 and 96 h, the suppression of HBsAg and HBeAg expression by 10-23 DNAzyme and LNAzyme in 2.2.15 cells were studied. Results The suppression of HBsAg and HBeAg expression by 10-23 DNAzyme and LNAzyme in 2.2. 15 cells were significant. The inhibitory effects caused by LNAzyme was more significant than that by thiolmodified 10-23 DNAzyme whose inhibitory effects were more significant than that of 10-23 DNAzyme. The inhibitory rates of LNAzyme and 10-23 DNAzyme thiolmodification reached （91.6±8.4）%, （78.4±2.0）% on HBsAg, respectivelly and （90.1±5.2）% , （76.4±4.8）% on HBeAg. The inhibitory effects of LNAzyme and thiolmodification of 10-23 DNAzyme were found 12 h after they were added to 2. 2.15 cells, and optimized at 48 h, effective inhibitory time for LNAzyme was 84 h, for thiolmodification 10-23 DNAzyme was 72 h. Addition of LNAzyme and 10-23 DNAzyme to 2. 2.15 cells didn＇t exert cytotoxicity. Conclusion 10-23 DNAzyme and LNAzyme have demonstrated significant inhibitory effects on the HBsAg and HBeAg expressions in 2. 2.15 cells. Morever, the inhibitory effects of LNAzyme is more significant than that of DNAzyme. LNAzyme is a specific anti-HBV therapeutic agent.