發展適合C型肝炎病毒感染之細胞培養模式

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

潘考祿(Kao-Lu Pan)

指導教授 ： 宋信文 ； 徐祖安

國立清華大學/工學院/化學工程學系/博士(2010年)

摘要

目前最先進的C型肝炎病毒體外培養系統就屬HCV-JFH1病毒株或是JFH1嵌合體的病毒株感染肝癌細胞株Huh7。對於JFH1以外的病毒株包括一般病人血清的HCV，都較難在Huh7細胞中複製與增殖。因此發展適合所有基因型HCV感染的細胞培養系統是非常重要的。一般最適合提供HCV感染的宿主細胞還是以具有高度分化的人類初代肝細胞 (human primary hepatocyte) 最佳，但是此類細胞的培養與功能的維持相當困難。因此本研究試圖利用幹細胞分化技術，分化間質幹細胞成為具有高度分化程度的肝臟細胞，以期能替代人類初代肝細胞成為HCV的宿主細胞。為此我們設計以白蛋白 (albumin；ALB) 與甲胎蛋白 (α-fetoprotein；AFP) 之調控元件為基礎，驅動表現分泌性鹼性磷酸酶白蛋 (secreted alkaline phosphatase；SEAP)，發展高效率細胞模式篩選平台可以篩檢具有肝分化潛能之小分子化合物，分別有Huh7-ePALB-SEAP和Huh7-ePAFP-SEAP兩株細胞。透過新穎小分子化合物的添加，誘導間質幹細胞走向高分化程度的肝臟細胞。分析系統的統計學參數Z' factor其值都大於0.5，表示適合進行高效率篩選程序。接著，我們進行小規模的化合物 (2000個化合物) 篩選。我們發現harmine和許多類固醇分別可以誘導ALB和AFP調控元件增加SEAP活性，可是卻不能有效地改進間質幹細胞的肝分化程度以及對HCV-JFH1的感受性。雖然尚未發現可以具肝分化潛能之小分子化合物，但此系統提供作為一個高效率的篩選平台，因此也不排除再篩選大量化合物庫的可能性。

另外，我們也設計一套可以有效監測JFH1病毒於細胞內活動的報導系統 (reporter system)，透過化合物篩選方式搜尋影響病毒生命週期的小分子化合物，例如抑制病毒活動和活化病毒生命週期的化合物。報導系統的設計概念是以HCV NS3/4A蛋白酶的作用機制為基礎，建構綠螢光蛋白 (enhanced green fluorescent protein；EGFP) 基因連接NS4B/NS5A序列與SEAP基因的報導基因，EG(△4B5A)SEAP，並轉染於Huh7.5細胞株產生Huh7.5-EG(△4B5A)SEAP。NS4B/NS5A連接胜肽是NS3/4A蛋白酶切割的位置，經過JFH1感染後可以使SEAP分泌到細胞外並方便被我們偵測。我們最佳化了此系統的分析條件，發現JFH1病毒於感染後第六天有較顯著性的SEAP活性產生，同時也利用已知的抗病毒藥物，分別對於病毒的進入 (entry)、複製 (replication) 和分泌 (secretion) 進行測試，都呈現出很好的抑制效果。系統的統計學參數Z' factor亦大於0.5，可作為高效率篩選的平台。對於發展抗病毒藥物以及應用於改進HCV細胞培養的條件，提供了一個穩健的分析方法。本研究亦利用此分析方法，小量分析39個類固醇化合物，發現triamcinolone acetonide (TA) 可以有效的增加細胞中HCV的NS3蛋白酶表現量和病毒基因組的複製量。TA或許可以提供作為改進HCV細胞培養的小分子化合物之ㄧ。

關鍵字

C型肝炎病毒 ； 白蛋白 ； 甲胎蛋白 ； 性鹼性磷酸酶白蛋 ； 報導系統

並列摘要

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.

並列關鍵字

Hepatitis C Virus ； albumin ； α-fetoprotein ； secreted alkaline phosphatase ； reporter system

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