Inhibition Of Hepatitis B Virus Expression And Replication By RNA Interference Technique

Objective: To observe the changes of hepatitis B virus replication as wellas expression by transfecting pGenesil-HBV X plasmid expressing smallinterfering RNAs (siRNA) that target HBV X gene region into HepG2.2.15cells , which at the basis of optimal dose ratio selection of polycationictransfection reagent METAFECTENE to pGenesil-EGFP . Methods: PartⅠ Selection of the optimal ratio . We designed differentratio of META [μl] to pGenesil-EGFP plasmid [μg] according to the Guidelinesbetween 2/1 and 7/1 , then cotransfected the META-plasmid complexes intocultured HepG2.2.15 cells . After 24 hours , the transfection rates weredetermined by using fluorescence microscope . Trypanblue test was performedto except the reductive possibility of cells' activation influenced by lipid . PartⅡ Transfecting the pGenesil-HBV X—META complexes toHepG2.2.15 cells , which performed as follows : (1) pGenesil-HBV X andliposome METAFECTENE complexes were made up according to PartⅠ, thencotransfected into HepG2.2.15 cells . After 24 , 48 , 72 hours , the levels ofHBV expression and replication in the supernatant were determined . (2) Thereplication levels of HBV-DNA were determined by fluorescence quantitativePolymerase Chain Reaction (PCR) . (3) The expression levels of HBsAg andHBeAg in the supernatant were determined with Time-resolvedImmunofluorometric Assay kit (TRFIA) ; the unrelated protein AFP in thesupernatant was determined with Microparticle ChemicluminescenceImmunoassay kit (MLIA) . (4) The expression of intracellular viral proteins wasdetermined by immunohistochemistry . Results: (1) The optimal ratio of METAFECTENE to pGenesil-EGFPplasmid was " 8μl:2.5μg " which could lead to the best rates of transfection . (2)After 24 hours of transfection , the expression of HBsAg and HBeAg in thesupernatant of transfected cells had no significant reduction ( P>0.05 ) ; at 48hours , TRFIA analysis showed that HBsAg and HBeAg were inhibited by28.47% and 38.67% (P<0.05); at 72 hours , the inhibitory rates were 32.16%and 42.86%(P<0.05). But the expression of AFP had no significant differenceat three time points . (3) Levels of HBV-DNA , a replication intermediate , alsodecreased by 44.88% and 45.9% at 48 hours and 72 hours respectively(P<0.05). (4) Immunocytochemistry revealed that the cells transfected withpGenesil-HBV X were dyed weakly or completely negative for detectableHBsAg levels . Conclusions: These results show that vector-based siRNA pGenesil-HBVX could efficiently suppress HBV antigen express and DNA replication inHepG2.2.15 cells , which indicated that RNA interference may be a valuabletool to control HBV infection .