中文摘要：

目的研究载乙型肝炎病毒（HBV）反义寡核苷酸的双重表面修饰肝实质细胞靶向阳性脂质体的基因转染，抗乙肝病毒作用和其介导基因转染的机制。方法以3β-[N-（N’，N’-二甲氨基乙基）一氨甲酰基]胆固醇（DC—Chol）和二棕榈酰磷脂酰胆碱（DPPC）为脂材，分别以谷甾醇葡萄糖苷（sito—G）和卞泽（Brij 35）为膜表面修饰成分，制备载HBV反义寡核苷酸的阳性脂质体。采用大鼠原代肝实质细胞和人肝癌细胞HepG 2．2．15，通过流式细胞分析、荧光显微镜观察和酶联免疫吸附试验（ELISA），考察脂质体对基因转染的促进作用及其病毒抑制作用；通过评价渥曼青霉素、尼日利亚菌素以及无涎胎球蛋白对其病毒抑制作用的影响，探讨其转染机制。结果以sito—G和Brij35对脂质体进行双重表面修饰，显著提高了脂质体的转染率和病毒抑制作用；荧光显微镜下观察到较强转染，反义寡核苷酸的胞内分布以在细胞核中为主；渥曼青霉素、尼13利亚菌素和无涎胎球蛋白均不同程度地降低了载反义寡核苷酸脂质体的病毒抑制作用。结论Brij35和sito—G双重修饰阳性脂质体显示出较高的基因转染效率和显著的病毒抑制作用，其基因转染过程以内吞和膜融合为主，并表现出肝实质细胞表面去唾液酸糖蛋白受体（ASGPR）的靶向选择性。

英文摘要：

Aim To study the transfection and anti-hepatitis B virus （HBV） effect of the co-modified hepatocytes-targeting cationic liposomes encapsulating anti-HBV antisense oligonucleotides （asON） , and to investigate the transfection mechanisms of the liposomes. Methods Dipalmitoylphosphatidylcholine （DPPC） and 3β- [ N- （ N＇, N＇-dimethylaminoethane） -carbamoyl ] cholesterol （DC-Chol） were used as the lipids, β-sitosterol-β-D-glucoside （sito-G） and Brij 35 were used to modify the liposomes. Flow cytometry （FCM）, fluorescence microscopy and enzyme-linked immunosorbent assay （ELISA） were utilized to evaluate the transfection improvement of the asON encapsulated in the liposomes in primary rat hepatocytes and the antigens inhibition activity in HepG 2.2. 15 cells. The transfection mechanisms were evaluated based on the influence of wortmannin, nigericin, and asialofetuin on the antigens inhibition in HepG 2.2.15 cells by ELISA. Results The co-modification with sito-G and Brij 35 significantly improved the transfection of the liposomes in primary rat hepatocytes and antigens inhibition effect in HepG 2. 2. 15 cells. Both transfection efficiency and antigens inhibition effect showed to be concentration-dependent with the asON-encapsulating liposomes. In fluorescence microscopy, the transfected cells showed strong fluorescence in primary rat hepatocytes, especially in the nuclei. Wortmannin, nigericin and asialofetuin decreased the antigens inhibition of the asON-encapsulating liposomes to different levels. Cationic liposomes modification with sito-G and Brij 35 could improve the transfection and antigens inhibition effect of the asON. The transfection mechanisms of the co-modified liposomes included endocytosis and membrane fusion. The ligand sito-G was confirmed to be able to enhance asialoglycoprotein receptor （ASGPR）-mediated endocytosis. Conclusion Co-modified hepatocytes-targeting cationic liposomes would be a specific and effective carrier to transfer asON into hepatocytes.