| Hepatitis C virus is an enveloped positive-strand RNA virus. It chronically infects 170 million people worldwide and more than 30-60 million people in China alone. About 50 percent of patients infected with HCV develop chronic liver diseases, as well as cirrhosis and hepatocellar carcinoma. Besides human beings, chimpanzee is the only animal susceptible to HCV infection. But ethical issues, difficult availability and the very high cost severely limit its use. Up to date, little progress has been made on HCV life cycle, pathological mechanism and screening antivirus drugs because of the absence of suitable animal and cell culture models.Mouse, widely used in medical research because of its easy availability, low cost and good productivity, is a typically small animal model. So mouse is an important candidate model for HCV infection. Thus, much effort had been taken to develop a mouse model, including engraftment of human liver fragment or hepatoma cell infected by HCV in vitro to severe combined immunodeficiency (SCID) mouse or athymic nude mouse. Until now, some successes had been achieved. HCV could be detected in mouse serum, but HCV replication was only taken place in the human liver cells or human hepatoma cells. The fundamental problems concerning viral replication have not been solved.Many HCV receptors, including human low density lipoprotein receptor, CD81, glycosaminoglycans (GAGs) and the human scavenger receptor class B type I (SR-BI), have been found through the study on the viral infectionmechanism. Besides that, the cell inner environmental factors also affect viral infection and replication. Submergence- induced protein-like factor (Sip-L) has been reported as a hepatic factor capable of supporting HCV replication in no permissive cells.HCV can not infect mouse, mouse liver cells or hepatoma cells. Were the molecular differences between human and mouse the main reason of mouse liver cells unsusceptible to HCV. Could they be infected by HCV when being transfected HCV receptors? In order to answer this question, we have done the following works.1. Construction of hLDLR eukaryotic expression vector. By RT-PCR, the two fragments of human LDLR cDNA (hLDLRK hLDLR2) were cloned from human hepatoma cell line HepG2 which can be infected by HCV. The hLDLRl was digested by restriction enzymes Hind III, Bgl II and the hLDLR2 with Bgl II, Xba I. The two DNA fragments purified from gel were inserted into the Hind III and Bgl II restriction sites of plasmid pcDNA3. After sequencing, we used the pcDNA3 with correct hLDLR sequences to transfect mouse hepatoma cells.2. Screening and identification of positive transfected cells. The recombined vector pcDNA3-hLDLR and pcDNA3-AlbEP-hCD81/hSip-L with mouse liver-specific albumin promoter, which had been constructed in our laboratory, were transfected into mouse hepatoma cell line Hepa 1-6 individually. After two weeks G418-selection, hCD81, hLDLR and hSip-L mRNA transcription were confirmed using RT-PCR. hCDSl proteins expression were confirmed using flow cytometric analysis (FACS) too. Because of a lack of anti-hLDLR and anti-hSip-L antibody, these two genes expression were confirmed on mRNA transcription only.3. Establishment of HCV infection model using the transgenic mouse hepatoma cells. To study whether these transgenic cells can be infected with serum derived HCV, cells were incubated for 24h at 37 with 200 LHCV-positive serum diluted in 1.8ml of medium without normal bovine calf serum. Cells were harvested on days 1, 3, 5, 7 post-infection. Analysis of HCV RNA in cells by RT-PCR demonstrated that in those cells stably expressing hLDLR, HCV plus-strand RNA were detected on days 1, 3, 5 and minus-strand RNA (intermediator that indicates HCV genome replication) were detected on day 5; In those cells stably expressing hCD81, HCV plus-strand RNA were detected on days 1, 3, 5 and minus-strand RNA were detected on day 3, 5; In those cells stably expressing hSip-L and the control Hepa 1-6 cell, plus-strand RNA were detected on day 1 and n...
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