载脂蛋白AI介导小干扰RNA靶向至肝脏的相关机制及抗HBV作用研究

HBV-infected patients are generally treated with interferon (IFN)-a and ribavirin which cannot clear the HBV in hepatitis cells. Recent years small interference RNA is becoming a new genetic therapeutics approach because of its stability and gene silence. But the systemic siRNA delivery technologies are important in vivo for its clinical application. As a drug delivery system the high-density lipoprotein (HDL) was focused now. But HDL particle contains hundreds of proteins and lipids so it is difficult to become a drug delivery technique for its clinical application. Our research team has finished the apolipoprotein A1(Apoa1) mediated liver targeting delivery of hydrophilic drugs under the support of China Nature Science Foundation. Recently it is reported that phospholipids and nucleic acid can combine to a steady spatial structure. So our team plans to form phospholipids and nucleic acid into HDL-siRNA, and study the Apoa1 mediated siRNA formation, structure and the nuclease's degradation effects using the new labeling methods and analysis techniques; how siRNA was release from HDL-siRNA complexes; whether it is mediated by SR-BI receptor and the cell signal triggered by receptor in molecular, protein and cell level; the liver targeting delivery of complexes and its inhibition effect to HBV virus. We hope to lay the foundation of siRNA delivery using Apoa1 as the carrier in vivo.

作为新型基因治疗手段，小干扰RNA（siRNA）为抗HBV基因治疗提供了新的策略,但体内递送体系成为制约的因素。HDL作为肝靶向载体受到关注，但存在三方面的问题：1）HDL本身成分复杂2）siRNA经过脂质或阳离子修饰，这些饰物会增加体内代谢负担。3）研究仅限于体内靶向性，而转运siRNA相关机制未见报道。因此本课题欲简化转运体系，以apoAI、卵磷脂和siRNA组成前体HDL-siRNA。利用新型标记方法和分析检测手段研究apoAI介导的siRNA组装、复合物组合方式及结构表征、是否免受核酸酶破坏；从分子、蛋白和细胞水平研究siRNA如何从复合物释放、进入受体细胞相关机制、涉及的相关酶、是否受SR-BI受体调控、受体调控所触发的信号通路；复合物体内靶向转运至肝脏效果、对HBV的抑制效果等，为apoAI作为siRNA体内靶向递送载体奠立基础。

本试验设计了针对HBV X区的siRNA序列, 考虑到siRNA合成成本及试验操作环境的要求，将编码siRNA的DNA 序列(DX)合成后克隆至载体GV115上，构建了siRNA的质粒载体。考察了DOTAP、DOPE、胆固醇对脂质体制备及表征的影响，进而考察apoAI对重组复合物制备及表征的影响，优化了复合物制备条件。以莹光染料罗丹明代替siRNA质粒，考察肝细胞对重组复合物和脂质体的摄取，结果表明apoAI可以促进对罗丹明的摄取吸收。体内分布表明肝脏部位染料积聚高于其他组织器官。体外药效表明细胞上清HBeAg和HBsAg的量明显低于对照组，PCR结果显示上清中HBVDNA明显低于对照组，表明设计的siRNA质粒载体明显抑制HBV转录和翻译。