Expression of CCAAT/enhancer-binding protein in cultured rat hepatic stellate cells and its significance / 中华肝脏病杂志

<p><b>OBJECTIVE</b>The expression of C/EBPalpha protein and mRNA during automatically activation process in primary cultures of HSCs were observed in order to explore its possible association with the proliferation and activation of HSCs.</p><p><b>METHODS</b>Immunocytochemistry, Western blot and RT-PCR were used to evaluated the expression of C/EBPalpha protein and mRNA; as well as the expression of alpha-SMA, Desmin, MMP2, type I procollagen (alpha1). The eukaryotic vector harboring the full length cDNA of C/EBPalpha was transfected into activated HSC, then immunocytochemistry was applied to confirm the transfection and evaluate the effect of transfection on the proliferation of HSC by calculating the PCNA-positive cells. The morphological changes of HSC were observed by use of phase-contrast microscope.</p><p><b>RESULTS</b>Constitutive expression of mRNA and protein of C/EBPalpha were detected in primarily cultured HSCs, and the protein was seen in both nuclei and cytoplasm with the latter being dominant. Their expression levels reached highest at day 2 of the culture, then decreased gradually when continually cultured to the day 4, 7, 10, on the other hand, the expression of alpha-SMA, MMP2 and ColI(alpha1) increased steadily. Transient transfection was verified by the fact that much more and stronger C/EBPalpha stain was observed in transfected HSCs than in void-vector transfected cells. In C/EBPalpha gene transfected HSCs, the number of PCNA-positive cells dramatically decreased compared with the void-vector transfected cells 24h after transfection. In addition, the C/EBPalpha gene transfected HSCs died 36 h after transfection, a few surviving cells became longer and thinner in morphology, however the void-vector transfected cells almost all remained alive.</p><p><b>CONCLUSIONS</b>C/EBPalpha was likely involved in the HSCs activation, and over-expressed C/EBPalpha by transfection had inhibitory influence on the proliferation of cultured rat HSCs.</p>

Effect of RAR-beta transfection on the proliferation and phenotype of rat hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the effect of RAR-beta transfection plus treatment with the corresponding ligand ATRA on the proliferation and phenotype of platelet-derived growth factor (PDGF)-activated hepatic stellate cells (HSC).</p><p><b>METHODS</b>PDGF-activated hepatic stellate cells of rats were transfected with eukaryotic expression vector pCMV-script-RAR-beta, which was verified by western blot. The proliferation of transfected HSC was assayed by BrdU incorporation as well as MTT methods. Their phenotype (alpha-SMA and desmin) was observed by immunocytochemistry assay with image analysis and RAR-beta protein expression was detected by western blot.</p><p><b>RESULTS</b>Transfection of RAR-beta gene and treatment with ligand ATRA could increase the expression of RAR-beta protein for at least 144h and inhibit the proliferation and the expression of alpha-SMA and desmin in PDGF-activated HSC. Significant statistical differences were perceived comparing with sham-transfected, only-PDGF treated, non-ligand treated and irrelevant ligand-treated HSC.</p><p><b>CONCLUSIONS</b>Transfected with RAR-beta gene as well as using related ligand ATRA could suppress the proliferation and reverse the activation phenotype of activated HSC.</p>