In Vivo Treatment Of HCV Core-Positive HepG2 Cells With The Transfer Of Recombinant Caspase-3 Treatment System Using 2'-5' Oligoadenylate Synthetase Gene Promoter

Objective:To investigate whether recombinant Caspase-3 treatment system using 2'-5'oligoadenylate synthetase (OAS) gene promoter can induce apoptosis specifically in HCV core-positive hepatocyte cells in vivo.Methods:Human hepatoma cell line HepG2 was transfected with pcDNA3.1-HCV-core-EGFP plasmid and selected by G418. Expression of HCV-core was detected by RT-PCR and immunocytochemistry. Parental HepG2 cells and HepG2 cells transfected with HCV-core were inoculated subcutaneously into syngenic Balb/c mice respectively. Tumor-bearing mice were treated by intratumoral injection of either pGL3-OAS-re-Caspase-3 or pcDNA3.1-re-Caspase-3(positive control) or pcDNA3.1 (negative control), which were wrapped by galactosylated chitosan-graft-polyethyleneimine(GC-PEI). Animals were sacrificed after 48 hours. The sections from the treated tumors were analyzed by immunohistochemistry for HCV-core, and immunofluorescence for Caspase-3 and HCV-core double staining. Apoptosis of HepG2 cells was estimated by TUNEL. Morphological Changes of apoptosis were observed by transmission electron microscope.Results:①HepG2/core cell line stably expressing HCV-core protein was established.②The animal model expressing HCV-core was established.③78±8% of the tumor cells expressed HCV-core protein in this animal model. The percentage of HCV-core and Caspase-3 co-expression was 76±6% in HepG2/core/pGL3-OAS-re-Caspase-3 group, 35±8% in HepG2/core/pcDNA3.1-re-Caspase-3 group,8±3% in HepG2/core/pcDNA3.1 group by double-label immunofluorescence. Apoptosis cells in HepG2/core/pGL3-OAS-re-Caspase-3 group (111±9/HP) and in HepG2/core/pcDNA3.1-re-Caspase-3 (122±10/HP) were significantly higher than that in HepG2/core/pcDNA3.1 group(10±2/HP) (P<0.05). And Apoptosis cells in HepG2/core/pGL3-OAS-re-Caspase-3 group (111±9/HP) was significantly higher than that in HepG2/pGL3-OAS-re-Caspase-3 group(13±5/HP) (P<0.05). The cells undergoing apoptosis in HepG2/core/pGL3-OAS-re-Caspase-3 group showed characteristic morphological features, including chromatin aggregation, nuclear and cytoplasmic condensation, partition of cytoplasm and nucleus into membrane bound-vesicles (apoptotic bodies).Conclusion:①pGL3-OAS-re-Caspase-3 treatment system can induce apoptosis specifically in HCV-core positive HepG2 cells in vivo.②The present results strongly suggest that the transfer of pGL3-OAS-re-Caspase-3 is an effective and promising gene therapy for HCV infection.