Background Activation and proliferation of hepatic stellate cells (HSC) is essentially involved in the development and progression of hepatic fibrosis. The most potent growth factor for HSC is platelet-derived growth...Background Activation and proliferation of hepatic stellate cells (HSC) is essentially involved in the development and progression of hepatic fibrosis. The most potent growth factor for HSC is platelet-derived growth factor receptor (PDGF) and PDGF receptor β subunit (PDGFR-β) is the predominant signal transduction pathyway of PDGF which is overexpressed in activated HSC. This study investigated the cleavage activity of hammerhead ribozyme targeting PDGFR-β mRNA in HSC and the effect on biological characteristics of HSC.Methods Expression vector of anti-PDGFR-β ribozyme was constructed and transfected into rat activated HSC with lipofectamin. The positive cell clones were gained by G418 selection. The expression of PDGFR-β, α-smooth muscle actin, and typeⅠand type Ⅲ collagen were detected by using Northern blot, Western blot and immunocytochemical staining, respectively. The cell proliferation was determined with MTT colorimetric assay. The cell apoptosis was analyzed by using flow cytometry, acridine orange fluorescence vital staining and transmission electron microscopy.Results The expression of PDGFR-β at mRNA and protein level was markedly reduced in ribozyme-transfected HSC by 49%-57% ( P <0.05-0.01). The proliferation and α-smooth muscle actin expression of ribozyme-transfected HSC were significantly decreased ( P <0.05-0.01), and the type Ⅰ and type Ⅲ collagen synthesis were also reduced ( P <0.01). In addition, the proliferative response of ribozyme-transfected HSC to PDGF BB was significantly inhibited. Otherwise, the apoptotic cells were significantly increased in ribozyme-transfected HSC ( P <0.01), and typical apoptotic cells could be found under transmission electron microscopy.Conclusions The anti-PDGFR-β ribozyme effectively cleaved the target RNA and significantly inhibited its expression, which blocked the signal transduction of PDGF at receptor level, inhibited HSC proliferation and collagen synthesis, and induced HSC apoptosis. These results suggest that inhibiting PDGFR-β expression of HSC may be a new target for the therapy of liver fibrogenesis, and ribozyme may be a useful tool for inhibiting PDGFR-β expression.展开更多
文摘Background Activation and proliferation of hepatic stellate cells (HSC) is essentially involved in the development and progression of hepatic fibrosis. The most potent growth factor for HSC is platelet-derived growth factor receptor (PDGF) and PDGF receptor β subunit (PDGFR-β) is the predominant signal transduction pathyway of PDGF which is overexpressed in activated HSC. This study investigated the cleavage activity of hammerhead ribozyme targeting PDGFR-β mRNA in HSC and the effect on biological characteristics of HSC.Methods Expression vector of anti-PDGFR-β ribozyme was constructed and transfected into rat activated HSC with lipofectamin. The positive cell clones were gained by G418 selection. The expression of PDGFR-β, α-smooth muscle actin, and typeⅠand type Ⅲ collagen were detected by using Northern blot, Western blot and immunocytochemical staining, respectively. The cell proliferation was determined with MTT colorimetric assay. The cell apoptosis was analyzed by using flow cytometry, acridine orange fluorescence vital staining and transmission electron microscopy.Results The expression of PDGFR-β at mRNA and protein level was markedly reduced in ribozyme-transfected HSC by 49%-57% ( P <0.05-0.01). The proliferation and α-smooth muscle actin expression of ribozyme-transfected HSC were significantly decreased ( P <0.05-0.01), and the type Ⅰ and type Ⅲ collagen synthesis were also reduced ( P <0.01). In addition, the proliferative response of ribozyme-transfected HSC to PDGF BB was significantly inhibited. Otherwise, the apoptotic cells were significantly increased in ribozyme-transfected HSC ( P <0.01), and typical apoptotic cells could be found under transmission electron microscopy.Conclusions The anti-PDGFR-β ribozyme effectively cleaved the target RNA and significantly inhibited its expression, which blocked the signal transduction of PDGF at receptor level, inhibited HSC proliferation and collagen synthesis, and induced HSC apoptosis. These results suggest that inhibiting PDGFR-β expression of HSC may be a new target for the therapy of liver fibrogenesis, and ribozyme may be a useful tool for inhibiting PDGFR-β expression.
