ABSTRACT
<p><b>OBJECTIVE</b>To develop HDV as a vehicle to deliver hammerhead ribozyme into hepatocytes, the effects of modified HDV was assessed on the activity of embedded hammerhead ribozyme in vitro and in vivo.</p><p><b>METHODS</b>In vitro activity of ribozyme or HDV-driven ribozyme was assessed by incubating with the [alpha-32 P]-ATP labeled HBV RNA substrates at different temperature. Huh-7 cells were cotransfected with ribozyme or HDV-ribozyme chimera and HBV genome, by which inhibition of ribozymes on HBV transcription in vivo were examined.</p><p><b>RESULTS</b>The results indicated that both temperature and secondary structure influenced the cleavage activity of HDV-driven ribozyme significantly. When the factors were eliminated, the HDV-driven ribozyme could act as well as its counterpart naked ribozyme. While in cultured cells the HDV-driven ribozyme had higher inhibition to HBV gene expression than that of ribozyme alone.</p><p><b>CONCLUSION</b>The results demonstrated that HDV may weaken the activity of embedded ribozyme in vitro, but make it enhanced in cultured cells. Thus, this study could provide a useful evidence to develop HDV as vector for liver-special delivery of ribozyme to against chronic HBV infection.</p>
Subject(s)
Humans , Base Sequence , Cell Line, Tumor , Genome, Viral , Hepatitis Delta Virus , Genetics , Molecular Sequence Data , Plasmids , Genetics , Protein Structure, Secondary , RNA, Catalytic , Chemistry , Genetics , Metabolism , RNA, Viral , Genetics , Metabolism , Structure-Activity Relationship , Substrate Specificity , Temperature , TransfectionABSTRACT
Overexpression of procollagen gene can cause the extraordinary increase of collagen's synthesis and therefore lead to the keloid and hypertrophic scar. To utilize ribozyme to suppress the expression of procollagen genes, a eukaryotic expression recombinant plasmid containing a dual-ribozyme gene against alpha 1 (I) and alpha 1 (III) procollagen genes was constructed. The ribozyme from in vitro transcription was incubated with target transcripts from recombinant plasmids which separately contained the fragments of the second exons of pro alpha 1 (I) and pro alpha 1 (III) collagen genes under various experimental conditions. The results showed that the dual-ribozyme could efficiently catalyze the specific cleavage of the target RNAs at 37 degrees C, 42 degrees C, 50 degrees C and Mg2+ concentration from 10 mmol/L to 20 mmol/L. This work provided a basis for further study on the ribozyme to suppress the expression of procollagen genes and control the cicatrization.