2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38791Chronic infection of hepatitis C virus (HCV) may lead to hepatic fibrosis and subsequently cirrhosis, although the underlying mechanisms have not been established. Previous studies have indicated that the binding of HCV E2 protein and CD81 on the surface of HSC may activate ERK/MAPK pathway to enhance the activate the transcriptional factor, AP-2. Such activation will lead to the increased protein level and activity of MMP-2, indicating that E2 may involve in the HCV-induced fibrosis. Our preliminary results indicated that the affect of E2 on the mRNA and protein levels of fibrosis-related molecules, including α-SMA, Collagen α (I), IL-6, IL-1β, TGF-β1, CTGF and MMP-2 all had effect and induced ROS reaction indicated that E2 may related with fibrosis process. Therefore, in this 3-year proposal, the functional domains of E2 gene responsible for E2-induced fibrosis will be identified by constructing plasmids containing E2 fragments. Meanwhile, a GST pull-down assay will be conducted to identify those molecules interacting with E2 and the involvement of identified molecules in the up-regulation of fibrosis-related molecules will be studied. During the 2nd year, further mutagenesis strategies, including site-directed mutagenesis and deletion mutation will be employed to further identify the vital region/residue for E2-induced fibrosis. Furthermore, the impact of E2 on ROS-related molecular pathway, such as JAK-STAT pathway, will be studied in this year. In the final year of this study, an in vitro co-culture system composed of a stable Huh7-E2 cell line and primary HSC will be established and analyzed to confirm the impact of E2 on fibrosis marker expression as seen in previous results and the possible impact of E2 on other fibrosis-related pathways, such as cellular senescence, will be investigated by analyzing the senescence markers (SA-β-gal and p16 staining) accumulation, p53 and Rb pathways of E2-expressing cells. Through this 3-year project, the mechanisms regarding to HCV-induced fibrosis will be further understood and more useful information may be provide for future intervention for chronic HCV infection.C 型肝炎病毒(HCV)的慢性感染會導致肝纖維化，進而造成肝硬化，但相關分子機制尚 未很清楚。有文獻指出HCV 套膜蛋白E2 與肝臟星狀細胞(HSC)膜上接受器CD81 結合 後，可藉由活化ERK/MAPK 的途徑活化轉錄因子AP-2，進而導致MMP-2 表現量及活 性上升，顯示E2 與HSC 的結合會造成HSC 活化而參與纖維化的過程。本研究室先前 研究發現，E2 對於HSCa-SMA、Collagen a (I)、interleukin (IL)-6、IL-1b、TGF-b1、 connective tissue growth factor (CTGF)、MMP-2 等纖維化因子的表現量皆會產生影響， 並進一步證實E2 可藉由刺激細胞內產生氧化性傷害使得TGF-b1 表現上升。為了更進 一步釐清E2 在纖維化過程中扮演的角色，此三年計畫的第一年中，將先針對E2 的結構 區域做深入的研究，除了探討與造成纖維化因子表現相關的結構區域之外，也進一步研 究可能與其結構區域作用的細胞內分子。第二年的計畫將探討E2 在氧化性傷害中可能 影響的相關路徑。在第三年中，將利用In vitro co-culture system 的模式，將穩定表現E2 的Huh7 肝癌細胞和HSC 進行共同培養，藉此模式來進一步驗證前兩年的研究成果。此 外，也將探討E2 對於纖維化其他機制的影響，例如對於senescence markers 的影響。希 望能藉由此計畫對與C 型肝炎相關的纖維化發生機制有更清楚的瞭解，如此將有助於瞭 解C 型肝炎病毒的致病機制，對於將來治療藥物的發展亦有幫助。C 型肝炎套膜蛋白E2纖維化氧化性傷害hepatitis C virusE2fibrogenesisoxidative damageProject