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研究生： 曾瓊瑩
Tseng, Chiung-Ying
論文名稱： 直接抗病毒藥物對慢性C型肝炎病人血漿脂肪的影響
Impacts of direct-acting antiviral agents on plasma lipid profiles of chronic hepatitis C patients
指導教授： 楊孔嘉
Young, Kung-Chia
學位類別： 碩士
Master
系所名稱： 醫學院 - 醫學檢驗生物技術學系
Department of Medical Laboratory Science and Biotechnology
論文出版年： 2016
畢業學年度： 104
語文別： 英文
論文頁數： 57
中文關鍵詞： 直接抗病毒藥物 、C型肝炎病毒 、脂肪代謝
外文關鍵詞： direct acting antiviral agent, hepatitis C virus, lipid metabolism
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    • C型肝炎病毒（Hepatitis C virus, HCV）造成了全球性的健康問題，現今大約有一億八千多萬人口受到C型肝炎病毒感染，且人數仍在持續增加中。C型肝炎病毒感染會引發急性和慢性肝炎的發生，並增加罹患肝硬化、肝癌、代謝紊亂和心血管疾病的風險。先前的研究已證實，C型肝炎病毒許多生長週期階段都有脂質的參與。C型肝炎病毒甚至可以調節宿主細胞中的脂質合成代謝，來維持病毒自身生命週期的穩定。在肝臟細胞中，C型肝炎病毒會抑制極低密度脂蛋白(Very-low-density lipoproteins, VLDL)的分泌，使脂質在肝組織中累積，提供有利於病毒複製與合成的環境。而在血液循環系統中，成熟的C型肝炎病毒顆粒會與極低密度脂蛋白和低密度脂蛋白(Low-density lipoproteins, LDL)等脂蛋白產生連結，並誘發極低密度脂蛋白和低密度脂蛋白之間的動態平衡失調，造成慢性C型肝炎病人的血脂代謝紊亂。目前，最能有效對抗C型肝炎病毒的治療藥物為直接抗病毒藥物(Direct acting antiviral agents, DAAs)。若在泛基因型C型肝炎病毒感染患者身上施以直接抗病毒藥物治療，其持續性病毒反應(Sustained virologic response, SVR)的成功率可高達90%以上。但是，直接抗病毒藥物治療是否能有利於改善慢性C 型肝炎(Chronic hepatitis C, CHC)患者的血脂代謝問題，仍然不清楚。因此，本研究想深入分析慢性C 型肝炎患者接受直接抗病毒藥物治療前後，其各類脂蛋白的變化。受試者為感染了病毒基因型1型的慢性C型肝炎患者，患者分別接受為期12周的安慰劑、Zepatier（Grazoprevir 與Elbasvir，為NS3/4A蛋白酶抑製劑與 NS5A抑製劑）和Harvoni（Ledipasvir與Sofosbuvir，為NS5A抑製劑與NS5B抑製劑）藥物治療。我們收集患者在治療前、治療期間1、4、8、12周和療程結束後第12周的血漿，將血漿連續進行兩次超高速密度梯度離心，分離出極低密度脂蛋白、低密度脂蛋白和高密度脂蛋白(High-density lipoproteins, HDL)。純化後的脂蛋白會進行三酸甘油脂(Triglyceride, TG)、膽固醇(Cholesterol, CHOL)和載脂蛋白(Apoprotein)的定量分析，用以評估直接抗病毒藥物治療對慢性C型肝炎患者脂肪代謝的影響。由此次的研究結果可看到，患者接受直接抗病毒藥物治療後，(i) 極低密度脂蛋白中的三酸甘油脂和膽固醇含量增加，這指出脂蛋白可從肝臟攜帶出較多的脂質分子至血液循環中，故脂肪累積在肝臟的現象可能得以緩解；(ii) 高密度脂蛋白的膽固醇含量增加，這表明心血管疾病的罹患風險降低；(iii) 極低密度脂蛋白中的三酸甘油脂相對於膽固醇的比例值上升，但低密度脂蛋白中的三酸甘油脂相對於膽固醇的比例值下降，這顯示三酸甘油脂的水解效率得到提升。由上述結果推斷，直接抗病毒藥物或許能扭轉C 型肝炎病毒所致的不正常脂肪代謝，並能減緩因C型肝炎病毒而誘發的脂肪肝、代謝紊亂和心血管疾病。

    Hepatitis C virus (HCV) is a global health problem that affects approximately 185 million people persistently. HCV infection causes acute and chronic hepatitis, and increases the risk for the development of liver cirrhosis, hepatocellular carcinoma, metabolic disorders and cardiovascular diseases. Previous researchers have reported that HCV life cycle is closely related with the host lipids. And even, HCV may regulate host cellular lipid homeostasis to maintain a stable viral life cycle. In liver, the very-low-density lipoprotein (VLDL) secretion is negatively modulated by HCV and leads to abnormal lipid accumulation in hepatocytes. In circulation, the mature HCV particles interact with lipoprotein, including VLDL and low-density lipoprotein (LDL). HCV infection is considered to induce the impairment of metabolic VLDL-to-LDL conversion and ultimately results in dyslipidemia. Currently, interferon-free direct acting antiviral agents (DAAs) treatment in pan-genotypic HCV-infected patients has achieved exceeding 90% sustained viral response (SVR). However, whether the DAAs treatment may benefit lipoprotein metabolism in chronic hepatitis C (CHC) patients remains elusive. To analyze lipoprotein changes in patients with HCV-genotype 1 infection receiving Zepatier (Grazoprevir/Elbasvir, NS3/4A protease inhibitor/NS5A replication complex inhibitor), Harvoni (Ledipasvir/Sofosbuvir, NS5A/NS5B inhibitor) and placebo, respectively, the following parameters were analyzed including triglyceride (TG) and cholesterol (CHOL) in plasma, VLDL, LDL and HDL in CHC patients at baseline, weeks at 1, 4, 8, and 12 of treatment and follow-up. In this research, the VLDL, LDL and HDL fractions from plasma were purified by two consecutive density-gradient ultracentrifugations. The results showed that the CHC patients with HCV-genotype 1 infection after DAAs treatment displayed that (i) the levels of VLDL-TG and VLDL-CHOL were increased, indicating that the relief of hepatic lipid accumulation; (ii) increment of HDL-CHOL, suggesting that the risk of cardiovascular diseases might be reduced; (iii) the TG/CHOL ratio was increased in VLDL not in LDL, displaying that an elevation of hydrolysis efficiency of TG. In conclusion, the anti-HCV DAAs might reverse the un-favoring lipid profiles and mitigate HCV-induced steatosis, metabolic disorders and cardiovascular diseases.

    Abstract （In Chinese） I Abstract （In English） III Acknowledgment V Index VI Table and figure index IX Abbreviations XI Items source XII Introduction 1 1. Molecular biology of hepatitis C virus (HCV) 1.1 The background of HCV 1.2 Life cycle of HCV 2. The clinical anti-HCV therapy 2.1 The development of HCV therapy 2.2 The DAAs in this research—Zepatier and Harvoni 2.2.1 Zepatier 2.2.2 Harvoni 3. Lipid metabolism 4. Relationship between HCV and lipid metabolism 4.1 HCV life cycle is closely related with the host lipids in hepatocytes 4.2 Changes of lipid metabolism in chronic hepatitis C (CHC) patients 5. Study goal Materials and Methods 7 1. Patients 2. Blood samples collection 3. VLDL, LDL and HDL isolation 3.1 Preparation of the centrifuge solutions 3.2 Two consecutive density-gradient ultracentrifugations 4. Quantification of apoAI and apoB by ELISA 5. Quantification of TG and CHOL by biochemical analysis 5.1 TG quantification 5.2 CHOL quantification 5.3 HDL-CHOL quantification 6. Formula for calculating the lipid recovery and lipid change ratio 7. Statistical analysis Results 12 1. Isolation of VLDL, LDL and HDL from plasma 2. Patient profiles 3. The HCV titers in the CHC patients were effectively reduced by Zepatier and Harvoni treatment 4. The lipid profile of CHC patient was improved after DAAs treatment (Zepatier treatment or Harvoni treatment) 4.1 Changes of TG and CHOL concentrations in longitudinal analysis of two CHC patients received 12-week Zepatier treatment after 16 weeks observation 4.2 After DAAs treatment, the levels of VLDL-TG, total CHOL, VLDL-CHOL, LDL-CHOL, HDL-CHOL gradually increased, but total TG level decreased 4.3 After DAAs treatment, the TG/CHOL ratio in VLDL gradually increased, but TG/CHOL ratio in LDL and plasma gradually deceased 4.4 After DAAs treatment, the apoB level in plasma and in VLDL and apoAI in VLDL gradually deceased Discussions 17 1. Comparison with previous clinical outcomes 2. The synthesis and secretion of VLDL negatively modulated by HCV were rescued and lipid loading efficacy of VLDL was increased in CHC patients receiving DAAs treatment 3. The lipid metabolism of VLDL and LDL was improved in CHC patients receiving DAAs treatment 4. HCV-induced imbalance in lipid homeostasis was reversed in CHC patients receiving DAAs treatment 5. HCV viral load had a direct effect of CHOL concentration in plasma and in various lipoproteins 6. Conclusion References 21 Tables and figures 25

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