Expression of exogenous rat collagenase in vitro and in a rat model of liver fibrosis

AIM: The present study was conducted to test thehypothesis that the introduction of the collagenase geneinto tissue culture cells and into a rat model of liver fibrosiswould result in the expression of enzymatically active product.METHODS: FLAG-tagged full-length rat collagenase cDNAwas PCR amplified and cloned into a mammalian expressionvector. NIH3T3 cells were then transiently transfected withthis construct. Expression of exogenous collagenase mRNAwas assessed by RT-PCR, and the exogenous collagenasedetected by Western blotting using anti-FLAG monoclonalantibodyEnzymatic activity was detected by gelatinzymography. To determine the effects of exogenouscollagenase production in vivo, the construct was boundto glycosyl-poly-L-lysine and then transduced into rats thathad developed liver fibrosis as a result of CCI4 plus ethanoltreatment. The hepatic expression of the construct and itseffect on the formation of liver fibrosis were demonstratedusing RT-PCR and immunohistochemistry.RESULTS: It was found that exogenously expressed ratcollagenase mRNA could be detected in NTH3T3 cellsfollowing transfection. Enzymatic ally active collagenase couldalso be detected in the culture medium. The recombinantplasmid was also expressed in rat liver after in vivo genetransfer. Expression of exogenous rat collagenase correlatedwith decreased deposition of collagen types I and Ⅲ in thelivers of rats with experimentally induced liver fibrosis.CONCLUSION: The expression of active exogenous ratcollagenase could be achieved in vitro and in vivo. It wassuggested that in vivo expression of active exogenouscollagenase may have therapeutic effects on the formationof liver fibrosis.

AIM:The present study was conducted to test the hypothesis that the introduction of the collagenase gene into tissue culture cells and into a rat model of liver fibrosis would result in the expression of enzymatically active product. METHODS:FLAG-tagged full-length rat collagenase cDNA was PCR amplified and cloned into a mammalian expression vector.NIH3T3 cells were then transiently transfected with this construct.Expression of exogenous collagenase mRNA was assessed by RT-PCR,and the exogenous collagenase detected by Western blotting using anti-FLAG monoclonal antibody.Enzymatic activity was detected by gelatin zymography.To determine the effects of exogenous collagenase production in vivo,the construct was bound to glycosyl-poly-L-lysine and then transduced into rats that had developed liver fibrosis as a result of CCl_4 plus ethanol treatment.The hepatic expression of the construct and its effect on the formation of liver fibrosis were demonstrated using RT-PCR and immunohistochemistry. RESULTS:It was found that exogenously expressed rat collagenase mRNA could be detected in NIH3T3 cells following transfection.Enzymatically active collagenase could also be detected in the culture medium.The recombinant plasmid was also expressed in rat liver after in vivo gene transfer.Expression of exogenous rat collagenase correlated with decreased deposition of collagen types Ⅰ and Ⅲ in the livers of rats with experimentally induced liver fibrosis.CONCLUSION: The expression of active exogenous rat collagenase could be achieved in vitro and in vivo. It was suggested that in vivo expression of active exogenous collagenase may have therapeutic effects on the formation of liver fibrosis.