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Background Transforming growth factor-β1 (TGF-β1) exerts strong fibrogenic potential in culture-activated HSCs Smad 4 is a key intracellular mediator for the transforming growth factor-β (TGF-β) superfamily of growth factors The aim of this study was to assess the effects of the antisense Smad 4 gene on Ito cell line, LI90 Methods The recombinant retroviral vector pLXSN-Smad 4 was constructed by cloning the rat antisense Smad 4 cDNA into the retroviral vector pLXSN Retroviruses with or without the antisense gene were obtained by transfecting pLXSN-Smad 4 and pLXSN vectors into PA317 cells Human hepatic stellate cells (HSCs) LI90 were infected with these retroviruses followed by selection with G418 The expression of Smad 4 was detected by Northern and Western blots Cell biological characteristics, including cell growth curve, 3H-TdR and 3H-proline uptake by HSCs and the production of extracellular matrix were assessed Results mRNA and protein expressions of Smad 4 in LI90 cells transfected with retrovirus containing the antisense Smad 4 gene were much lower than those in LI90 cells transfected with empty vector or parental LI90 cells Cells hypoexpressing the Smad 4 gene exhibited a slower rate of growth, a lower uptake of 3H-TdR and 3H-proline ( P <0 01), and smaller production of th extracellular matrix, compared with parental LI90 cells and cells transfected with empty retrovirus Conclusions The antisense Smad 4 gene can suppress the expression of the Smad 4 gene, reduce endogenous production of Smad 4 mRNA and protein, block TGF-β1 signaling pathway, inhibit activation of Ito cells, obstruct the growth of Ito cells, decrease the production of the extracellular matrix (ECM) Our results may provide a basis for the development of antifibrotic gene therapy
Background Transforming growth factor-β1 (TGF-β1) exerts strong fibrogenic potential in culture-activated HSCs Smad 4 is a key intracellular mediator for the transforming growth factor-β (TGF-β) superfamily of growth factors The aim of this study was to assess the effects of the antisense Smad 4 gene on Ito cell line, LI90 Methods The recombinant retroviral vector pLXSN-Smad 4 was constructed by cloning the rat antisense Smad 4 cDNA into the retroviral vector pLXSN Retroviruses with or without the antisense gene were obtained by transfecting pLXSN-Smad 4 and pLXSN vectors into PA317 cells Human hepatic stellate cells (HSCs) LI90 were infected with these retroviruses followed by selection with G418 The expression of Smad 4 was detected by Northern and Western blots Cell biological characteristics, including cell growth curve, 3H -TdR and 3H-proline uptake by HSCs and the production of extracellular matrix were judged Results mRNA and protein expressions of Smad 4 in LI90 cells transfected with retrovirus containing the antisense Smad 4 gene were much lower than those in LI90 cells transfected with empty vector or parental LI90 cells Cells hypoexpressing the Smad 4 gene express a slower rate of growth, a lower uptake of 3H-TdR and 3H-proline (P <0.01), and smaller production of extracellular matrix, compared with parental LI90 cells and cells transfected with empty retrovirus Conclusions The antisense Smad 4 gene can suppress the expression of the Smad 4 gene, reduce endogenous production of Smad 4 mRNA and protein, block TGF-β1 signaling pathway, inhibit activation of Ito cells, obstruct the growth of Ito cells, decrease the production of the extracellular matrix (ECM) Our results may provide a basis for the development of antifibrotic gene therapy