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標題: | 中草藥SBEL1對C型肝炎病毒生活史的多重效用 Multiple Effects of Chinese Herbal Medicine SBEL1 on Hepatitis C Virus Life Cycle |
作者: | Cheng-Wei Lin 林政緯 |
指導教授: | 余明俊(Ming-Jiun Yu) |
關鍵字: | 中草藥,抗病毒劑,C型肝炎病毒,C型肝炎病毒生活史,非結構性蛋白5A, Chinese herbal medicine,antiviral agent,HCV,HCV life cycle,Nonstructural protein 5A, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | C型肝炎病毒(Hepatitis C virus, HCV) 在全世界估計感染1億八千五百萬人,慢性感染可導致肝纖維化、肝硬化以及肝細胞癌。HCV 的治療方式已大幅進步並可治癒廣泛病患,但治療價格昂貴。為找尋價格便宜的替代治療,我們對中草藥公司在台灣有種值的六種中草藥(編號SBEL1到6)進行篩選。相較於對照組,SBEL1被發現抑制90%的HCV活性。此篇研究探討SBEL1對於HCV生活史的影響並分離SBEL1的活性成分。Immunoblotting的結果顯示SBEL1的前處理顯著降低HCV非結構蛋白3的量23%相較於對照組,表示SBEL1阻擋HCV進入細胞。在Huh7.5.1細胞中, SBEL1顯著降低39%的internal ribosome entry site (IRES) 驅動的螢光活性,顯示SBEL1 對於IRES驅動轉譯活性的影響。在HCV感染的Huh7.5.1細胞中,SBEL1顯著降低78%的HCV的RNA量,表示SBEL1對於HCV RNA複製的影響。SBEL1顯著降低非結構蛋白5A(NS5A)在第235個氨基酸serine的磷酸化,此serine的磷酸化在先前研究指出和HCV複製有關。利用活性炭的色層分離,我們得到四個分層,並發現SBEL1中活性炭不吸附的分層抑制HCV的活性但不影響細胞存活。因此,我們的發現顯示SBEL1藉由降低HCV的進入、IRES驅動的轉譯活性、HCV的RNA複製以及NS5A的磷酸化達到對於HCV生活史的多重抑制效果。此外,利用活性炭色層分離,我們可以分離SBEL1中抑制HCV活性的分層與細胞毒殺的分層。 Hepatitis C virus (HCV) infects an estimated 185 million people. Chronic infection can lead to liver fibrosis, cirrhosis and hepatocellular carcinoma. HCV therapy has been rapidly improved and cures vast patients, but at a high cost. To seek for alternative HCV treatments with low cost, we screened six Chinese herbal medicines (CHMs, numbered SBEL1 to 6) already grown by herbal medicine companies in Taiwan. SBEL1 was found to inhibit HCV activity by 90% compared to the vehicle control. This study investigated the effects of SBEL1 on HCV life cycle and aimed to isolate active compound(s) of SBEL1. Immunoblotting results showed that SBEL1 pretreatment reduced NS3 abundance in HCV-infected Huh7.5.1 cells by 23%, suggesting that SBEL1 blocks HCV entry. SBEL1 significantly reduced internal ribosome entry site (IRES)-driven luciferase reporter activity in Huh7.5.1 cells by 39%, indicating an impact of SBEL1 on IRES-mediated translation activity. In HCV-infected Huh7.5.1 cells, SBEL1 was found to significantly reduce HCV RNA levels by 78%, consistent with an effect of SBEL1 on HCV replication. SBEL1 reduced phosphorylation of the non-structural protein 5A (NS5A) at the serine 235, which was previously shown with a strongly correlation with HCV replication. With activated charcoal separation, we obtained four fractions and found that charcoal unbound fraction of SBEL1 inhibits HCV reporter activity without effects on cell viability. Our observations are compatible with multiple inhibitory effects of SBEL1 on HCV life cycle by reducing HCV entry, IRES-mediated translation, HCV replication and NS5A phosphorylation. Moreover, with the activated charcoal separation, we were able to separate HCV-inhibitory fraction from cytotoxic fractions of SBEL1. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18462 |
全文授權: | 未授權 |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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