ABSTRACT. We investigated the effects of hepatitis B virus (HBV) S/C double gene loci antisense locked nucleic acid on replication and expression of HBV in hepatitis transgenic mice. HBV mice (N = 30) were randomly divided into five groups of six mice: 5% glucose solution control, empty liposome control, single-target S, single-target C, and dual-target SC groups. An antisense locked nucleic acid fragment was injected into the mice. Serum HBsAg, serum HBV DNA, HBV C-mRNA expression in liver tissue, HbsAg and HbcAg expression in hepatocytes, serum albumin, alanine transaminase (ALT), urea nitrogen, and creatinine were detected. Liver and kidney sections were examined for the effects of antisense locked nucleic acid. The expression of HBsAg was markedly inhibited; the inhibition rates of the S, C, and SC target groups were 36.63, 31.50, and 54.87%, respectively; the replication of HBV DNA was also inhibited: 23.97, 21.13, and 35.83%, respectively. After injection at 1, 3, and 5 days, the corresponding rates for HBsAg inhibition were 14.40, 25.61, and 31.33%, and for HBV DNA inhibition they were 11.04, 19.24, and 24.13%. Compared with the control group, the differences in serum albumin, ALT, urea nitrogen, 10087-10095 (2015) and creatinine in each group were not statistically significant, and the number of HbsAg-and HBcAg-positive cells in the mouse liver was significantly reduced. The liver and kidney tissues were normal. The gene therapy had significant inhibitory effects on the replication and expression of HBV in transgenic mice, and double-gene targeting was better than single-gene targeting.
ABSTRACT. The aim of this study was to investigate the effects of inhibition of the hepatitis B virus (HBV) S gene by polypurine region locked nucleic acid on viral replication in cells. We designed and synthesized a locked nucleic acid, phosphorothioate oligonucleotides, unmodified oligonucleotides, and unrelated control sequence for the hepatitis B virus S gene polypurine region. HepG2.2.15 cells were transfected by cationic liposome, and fluorescence quantitative polymerase chain reaction technology (PCR) and time-resolved fluoroimmunoassay technology was utilized to monitor the content of HBV DNA, HbsAg, and HBeAg at 2, 4, 6, 8 and 10 days post-transfection. The effects on cell metabolism were detected by four methyl thiazolyl tetrazolium assay. The locked nucleic acid had an obvious effect on HBV DNA replication and HBsAg and HBeAg expression in a dose and time dependent manner. The inhibition rates were 52.14, 57.48, and 29.63% after 6 days, respectively. The locked nucleic acid had no significant effect on cell metabolism. The HBV S gene polypurine region locked nucleic acid could effectively inhibit the replication of HBV in vitro, and could pro- 5446©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (2): 5445-5451 (2015) vide an effective target for the treatment of HBV and a theoretical and experimental basis for anti-gene therapy.
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