Development of a Cell Culture Model for Hepatitis C Virus (HCV) Infection

• Main Authors: Pan, Kao-Lu, 潘考祿
• Other Authors: Sung, Hsing-Wen
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/28740647353985454042

博士 === 國立清華大學 === 化學工程學系 === 98 === To date, the most advanced system for studying HCV infection and replication is to employ the JFH1 clone and/or its derivative JFH1-chimera combined with HCV-permissive cell lines, such as Huh7 or Huh7.5 cell lines. However, Huh7 or Huh7-derived cells were known to be non-ideal hosts to permit propagation of HCV virion obtained from HCV-positive patients. Therefore, it is important to develop cell culture systems for the infection/propagation of various HCV genotypes. In general, human primary hepatocytes are the most suitable host cells for HCV infection but they are difficult to be acquired from human bodies and also difficult to be maintained in cell culture. In this study, we attempted to obtain highly differentiated human hepatocytes derived from human mesenchymal stem cells (MSCs). The ultimate goal of this project is to provide a platform technology so that human primary hepatocytes can be substituted by MSCs-derived hepatocytes for studying HCV in cell culture. In order to reach this goal, we first attempted to create two cell-based reporter systems based on gene expression of albumin and α-fetoprotein to screen for compounds with potentials to enhance hepatic differentiation. We constructed two novel reporter genes, i.e., ePALB-SEAP and ePAFP-SEAP, wherein the expression of the secreted alkaline phosphatase (SEAP) is under the control of the regulator element of albumin (ALB) and a-fetoprotein (AFP) gene respectively. These two reporter genes were transfected into Huh7 cells and such cells, i.e., Huh7-ePALB-SEAP and Huh7-ePAFP-SEAP, were then employed to screen compounds with the capability of up-regulating the ALB or AFP regulator elements using SEAP as the reporter enzyme. The Z' factors in these screening systems were > 0.5 indicating that both systems are suitable for high-throughput screening of prospective ALB- or AFP-inducible compounds. Few compounds were found to be able to enhance the SEAP activity. However, these few compounds were identified to be false positives because they could not increase the levels of albumin or fetoprotein expression in human MSCs-derived hepatocytes. Therefore, we attempted to create an alternative reporter system for directly monitoring HCV infection/replication. The new reporter cell line, Huh7.5-EG(△4B5A)SEAP, was created. The reporter gene, EG(△4B5A)SEAP, encodes a viral protease-cleavable fusion protein in which the enhanced green fluorescence protein (EGFP) is linked to SEAP in frame via △4B5A, a peptide cleavage substrate for NS3/4A viral protease. Our studies demonstrate that virus replication/infection in Huh7.5-EG(△4B5A)SEAP cells can be indirectly quantified by measuring the SEAP activity in cell culture medium. The Huh7.5-EG(△4B5A)SEAP cells were also shown to be a suitable reporter cell line for understanding the intercellular activity of HCV by using known compounds targeting multiple stages of HCV life cycle. This system can be a robust assay for discovering key factors, such as compounds, cytokines, and microenvironment, to improve HCV-permissiveness of hepatocytes. This system was employed to analyze a small compound library contsisting of 39 steroid compounds. The results show that the amount of intercellular NS3 protease and HCV RNA can be effieiently enhanced by the administration of triamcinolone acetonide (TA) in hepatoma cell line. We suggest that TA could be a key factor to improve HCV-permissiveness of hepatocytes.