Effect Of Ribozyme Targeting Platelet-derived Growth Factor Receptor βSubunit Gene On Biological Characteristics Of Hepatic Stellate Cell

Background and designHepatic fibrosis and liver cirrhosis are common pathological consequences by various chronic liver diseases which may show a continuation or repeated exacerbations of inflammation, necrosis and degeneration. Such pathological processes are associated with too much extracellular matrix deposition in the liver and impediment to liver regeneration, eventually leading to hepatic cirrhosis. The hepatic stellate cells (HSC) are activated by some soluble cytokines and altered composition of local extracellular matrix during the process of repairement of liver injury. The activated HSCs are the major sources of the collagens, as well as of the tissue inhibitors of metalloproteinases (TIMPs) which inhibit collagen degradation. Therefore, the activation and proliferation of HSC is the key procedure during the hepatic fibrosis.Platelet-derived growth factor (PDGF) is a kind of cytokines secreted by platelet, Kupffer cell, sinusoidal endothelial cells and activated HSC, which has two homologous polypeptides, the PDGF-A chain and the PDGF-B chain to form disulfide-linked dimers AA, AB and BB. During the hepatic fibrosis, the chain B of PDGF is overexpressed in the fibrotic septa and area of inflammatory necrosis. PDGF-BB is a more potent stimulant for HSC proliferation and migration, so PDGF plays a key role in the development of hepatic fibrosis.Although PDGF is the fierce mitogen of HSC, its effect depends on the expression of a specific dimeric transmembrane receptor, composed of a and P subunits to form act, ap, or pp. The PDGF a-receptor subunit binds all PDGF isoforms, whereas the P-receptor binds PDGF-BB with high affinity and PDGF-AB with lower affinity, but does not bind PDGF-AA. PDGF receptor-p subunit is not expressed in quiscent HSC or normal liver and do not response to PDGF, but is overexpressed in activated HSC or in injured liver and synchronized with the proliferation of HSC, which suggests that PDGF receptor-p subunit be closely related to the proliferation of HSC. Recent studies demonstrated that PDGF receptor-p is the major signal transduction pathway of PDGF in HSC (the activated HSC mainly expresses PDGF receptor-p) . PDGF-B chain leadsproliferation and migration of HSC by interaction with its membrane PDGF receptor-p and activation of phosphatidylinositol 3-kinase.Ribozyme is a kind of small catalytic RNAs capable of promoting the specific cleavage of target-RNA, and has provided a new and special stool for gene therapy. To our knowledge, there was no report which use ribozyme technologies to cleave the PDGF receptor p subunit in the study of liver fibrosis. In this study, hammerhead ribozyme genes targeting the PDGF receptor-P subunit extracellular domain were designed and synthesized, and its expression vector was constructed and transfected to HSC cultured in vitro using lipofectamin. In the meanwhile, we investigated the effect of anti-PDGF receptor p subunit ribozyme on biological characteristics of HSC and the role of PDGF receptor P subunit during the development of hepatic fibrosis, in order to find a new pathway in the treatment of hepatic fibrosis.MethodsThe model of hepatic fibrosis in rat was made by injection of carbon tetrachloride. PDGF receptor-p subunit, collagen I, collagen III and a-SMA in hepatic tissue during the hepatic fibrosis were detected with imrnunohistochemistry. The correlations between PDGF receptor-p subunit and collagen I, III and a-SMA were analyzed by software of SAS after the results of immunohistochemistry were semi-quantified.A 609bp of PDGF receptor-p subunit cDNA fragment of rat was amplified by reverse-transcription PCR (RT-PCR), then cloned into T-vector under the control of T7 promoter, named pPDGFR-p. The ribozyme genes, targeting the PDGF receptor-p subunit, RZ1 and RZ2, were designed according to the analysis of the secondary structure of PDGF receptor-p subunit mRNA with computer. The two hammerhead ribozyme genes (RZ1, RZ2) were cloned into vector pi.5 between 5'-cis ribozyme and 3'-cis ribozyme, named pRZl and pR...