通过受体介导法将核酶特异导入肝细胞以阻断HBV蛋白表达的初步研究

Preliminary study on the inhibition of hepatitis B viral expression in vitro via specific receptors mediated ribozyme transfer

应用半乳糖末端糖蛋白受体（ＡＳＧＰ－Ｒ）介导的内吞作用，将外源基因导入真核细胞，与脂质体介导的转染和细胞表面转铁蛋白受体（Ｔｆ－Ｒ）介导的内吞作用相比，虽然三种方式均能有效介导外源基因的转移，但ＡＳＧＰ－Ｒ法具有肝细胞特异性，而脂质体法和Ｔｆ－Ｒ法不具此特性。将克隆于真核表达载体的针对乙型肝炎病毒（ＨＢＶ）ｍＲＮＡＰｒｅＣ／Ｃ区的核酶质粒ｐＣＭＶ－Ｒｉｐｃ特异性导入肝细胞并发挥作用，通过酶联免疫吸附法（ＥＬＩＳＡ）检测细胞培养液中的乙型肝炎表面抗原（ＨＢｓＡｇ）和ｅ抗原（ＨＢｅＡｇ），评价核酶在细胞水平对ＨＢＶ抗原表达的阻断作用。结果表明当核酶质粒ｐＣＭＶ－Ｒｉｐｃ与ＨＢＶ抗原表达质粒ｐＵＣ－２ＨＢＶ共转染ＨｅｐＧ２细胞时，核酶对ＨＢｓＡｇ和ＨＢｅＡｇ表达的抑制率分别为５５．２９％和６８．７３％。

Comparing with liposome and transferrin receptors (Tf-R) mediated transfection, a plasmid harboring foreign gene with hepatic specificity could be transfected via asialoglycoprotein receptors (ASGP-R) mediated endocytosis. A recombination plasmid pCMV-Ripc harboring a hammerhead ribozyme which aim at the pregenome RNA of HBV ayw subtype at the 1796 site in the genome was delivered into HepG2 cells (HBV transient transfected system) and 2.2.15 cells (HBV permanent transfected system) via receptors mediated gene transfer methods. ELISA results showed that the inhibition of HBsAg and HBeAg expression is 55.29% and 68.73% respectively when pCMV-Ripc is co-transfected with pUC-2HBV in HepG2 cells via ASGP-R mediated DNA transfer method. The result indicated that pCMV-Ripc is efficient to inhibit the expression of HBV antigen and ASGP-R method is a safe, liver-targeted delivery system, therefore it is possible to develop an in vivo gene therapy strategy aiming to the treatment of hepatic infectious disease.