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目的在细胞水平上探讨10-23脱氧核酶(10-23DRz)成为新型乙型肝炎基因治疗药物的可能性。方法设计合成针对乙型肝炎病毒(HBV) ayw1亚型前C/C区的2031位点的10-23DRz,并对其进行硫代磷酸化修饰,同时针对该位点设计一条未硫代化修饰的10-23DRz,经脂质体转染HepG22.2.15细胞(简称2.2.15细胞)中观察其对HBV基因表达的抑制效应。结果修饰与未修饰的10-23DRz作用于2.2.15 细胞后均可显著抑制乙型肝炎表面抗原(HBsAg)和乙型肝炎e抗原(HBeAg)的表达,最高抑制率分别可达93.75%和90.26%,有效抑制持续时间可达96 h,修饰后的脱氧核酶在细胞内对HBsAg和HBeAg表达的抑制作用时间长于未修饰的DRz,其抑制率的最高值低于未修饰的DRz,但两者明显高于作为对照的反义脱氧寡核苷酸。对2.2.15细胞内HBV DNA复制无明显影响,未见明显的细胞毒性作用。结论经过硫代化修饰的10-23DRz和未修饰的10-23DRz在2.2.15细胞模型中能高效阻断HBV DNA的表达,是一种高度特异性的、高效的基因治疗剂。
Objective To investigate the possibility of 10-23 DNAzyme (10-23DRz) becoming a novel gene therapy drug for hepatitis B at the cellular level. Methods The 10-23DRz of 2031 site in pre-C / C region of hepatitis A virus (HBV) ayw1 subtype was designed and synthesized, and its phosphorylation was modified by thiophosphorylation. At the same time, a non-thiolated modification Of 10-23 DRz, HepG22.2.15 cells (referred to as 2.2.15 cells) were transfected with liposome to observe the inhibitory effect on HBV gene expression. Results Both modified and unmodified 10-23DRz inhibited the expression of HBsAg and HBeAg in 2.2.15 cells with the highest inhibitory rates of 93.75% and 90.26 %, And the effective inhibition time can reach 96 h. The inhibitory effect of modified DNAzyme on HBsAg and HBeAg expression in cells is longer than that of the unmodified DRz, the highest inhibition rate is lower than the unmodified DRz, but two Were significantly higher than the control antisense oligonucleotide. 2.2.15 intracellular HBV DNA replication had no significant effect, no significant cytotoxic effects. Conclusion The 10-23DRz modified by thiolation and the unmodified 10-23DRz can block the expression of HBV DNA efficiently in 2.2.15 cell model, which is a highly specific and efficient gene therapy agent.