Hammerhead Ribozyme-mediated Inhibition Of Connective Tissue Growth Factor In Liver Fibrosis Induced By Hepatic Stellate Cells In Vitro

Hepatic fibrosis is a common response to chronic liver injury from many causes,which are characterized by excessive extracellular matrix (ECM) deposition. Patients with various types of liver diseases will develop advanced hepatic fibrosis and liver cirrhosis or even liver cancer, so hepatic fibrosis is considered to be a common pathway to various kinds of chronic liver diseases to deteriorate.. Reversibility of hepatic fibrosis has been reported both experimentally and clinically. However, no effective method of treatment is available until now. It's therefore that a safe and effective method is expected to develop for treating hepatic fibrosis.The hepatic stellate cell (HSC) is the key fibrogenic cell of the liver and represents a paradigm cell type in studies of the pathogenesis of tissue fibrosis. After any cause of liver injury, quiescent HSCs undergo activation to proliferative, fibrogenic, and contractile myofibroblasts with increased expression ofα-smooth muscle actin (α-SMA). The activated HSCs can synthesize almost all of extracellular matrix (ECM) in liver. The HSC is thought to be a target cell type for the design of anti-hepatic fibrosis.Connective tissue growth factor (CTGF) plays an important role in liver fibrogenesis. Firstly, CTGF as a down-stream mediator of TGF-βinduces production of collagen type I in HSC and pancreatic stellate cells. Secondly, both CTGF and TGF-βare over-expressed in various fibrotic diseases. TGF-βis expressed in the early stage of fibrogenesis and CTGF is thought as a major player in chronic fibrotic disorder. Thirdly, CTGF is mainly expressed in mesenchymal cell and its action is limited to connective tissue. For these reasons targeting CTGF to block hepatic fibrosis opens up new vistas of research for scientists.Ribozyme is a small molecule nucleic acid (RNA) with enzyme cutting activity. The specific target RNA is recognized and binded by the specific sequence of ribozyme through complementary base, then substrate RNA molecules is cleaved and target gene expression is inhibited. Ribozyme with the unique advantages of sequence specificity, not encoding protein, no immunogenicity and being reused, is favored in the field of gene therapy. The hammerhead ribozyme is more effective, because of its folding structure of RNA easily to be dissociated. The mode which plays trans-form cleavage activity easily to be designed, together with a low requirements to the target sequence, and with a little cost, the hammerhead ribozyme has been successfully used in many organisms including bacteria, plants and animals.Two sources of ribozyms are available: direct chemical synthesis or transcription from a recombinant vector including ribozyme cDNA sequence. In this study a hammerhead ribozyme targeting human CTGF mRNA at 896 base was designed with ribozyme design software. On both flanks of the hammerhead ribozyme were added a self-cutting enzyme ie: a 5'-cis-Rz and 3'-cis-Rz to reduce conformational change of hammerhead ribozyme and increase hammerhead ribozyme activity and decrease mismatch between ribozyme and substrate.In this study lipofectamine 2000, a kind of liposomes, was used as transfer vector. Liposomes, which characterized as simply preparation, fewer side effects, no immunogenicity and a large carrying quantity, can carry exogenous gene though its electric effect. Recombinant plasmid pTriEx2-Rz containing CTGF hammerhead ribozyme cDNA sequence was constructed and transfected into human hepatic stellate cells (LX2). Empty plasmid pTriEx2 as control. The cutting role of hammerhead ribozyme on CTGF mRNA in LX2 was analysed by RT-PCR. TGFβ1 was added in the medium of the LX2 transfected with pTriEx2-Rz and cultured for 24h. Collagen type I secretion was measured using ELISA method. Flow cytometry was used to identify the change of LX2 cycle followed the LX2 transfected with pTriEx2-Rz.The results are as follows:1,The recombinant plasmid pTriEx2-Rz was constructed successfully, which proved by double enzyme digestion and sequencing test.2,Compared with empty plasmid pTriEx-2, both CTGF mRNA and collagen type I mRNA decreased significantly in the LX2 transfected by pTriEx-2-Rz (p<0.05); Compared with empty plasmid pTriEx-2 under the stimulation of TGF-β1, eithor CTGF mRNA or collagen type I mRNA was decreased significantly in the LX2 transfected by pTriEx-2-Rz under same stimulation of TGF-β1 (p<0.05).3,Compared with LX2 transfected with pTriEx-2, the collagen type I secretion decreased significantly in the LX2 transfected with pTriEx-2-Rz (p<0.01); The LX2 transfected with pTriEx2-Rz under the stimulation of TGF-β1 showed lower level of collagen type I secretion than that of the LX2 transfected with pTriEx-2 under the stimulation of TGF-β1.(p<0.01).4,LX2 transfected with pTriEx2-Rz induce an increase of the cell number in the phage of G0-G1 and decrease of cell number the number, suggestting that hamhead ribozyme targeting CTGF mRNA can decrease synthesis of DNA in the LX2 and further inhibit proliferation of the cells.The results show that:The recombinant plasmid pTriEx2-Rz containning CTGF hammerhead ribozyme cDNA sequence show a property of cutting CTGFmRNA in LX2. The pTriEx2-Rz are able to inhibite collagen type I mRNA transcription and protein secretion after being transfected into LX2. Furthermore, the DNA synthesis of LX2 can be inhibited by PTriEX2-Rz and the proliferation of LX2 was decreased accordingly. The transcription of CTGF mRNA and collagen type I mRNA as well as the secretion of collagen type I are promoted by exogenous TGF-β1;CTGF mRNA synthesis was partially blocked in the LX2 that transfected with recombinant plasmid pTriEx2-Rz , which can inhibit the effect of TGF-β1 on LX2-mediated fibrosis.These data support a role for CTGF in HSC-mediated fibrogenesis and develop a new method in which hamhead ribozyme targeting CTGF to block HSC-mediated fibrosis and highlight CCN2 as potential novel therapeutic targets.