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系統識別號 U0026-1501201218073300
論文名稱(中文) 建立 C 型肝炎病毒感染之報導系統
論文名稱(英文) Establishment of a Reporter System for Hepatitis C Virus Infection
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 100
學期 1
出版年 101
研究生(中文) 蔡佩汝
研究生(英文) Pei-Ju Tsai
學號 t36984019
學位類別 碩士
語文別 中文
論文頁數 123頁
口試委員 指導教授-楊孔嘉
口試委員-張定宗
口試委員-王憲威
口試委員-李景欽
中文關鍵字 JFH1-SEAP 細胞株  C型肝炎病毒基因型 2a  分泌性鹼性磷酸酶報導系統 
英文關鍵字 JFH1-SEAP cell  HCV genotype 2a  secreted alkaline phosphatase reporter 
學科別分類
中文摘要 C型肝炎病毒之正單股RNA能轉譯出三種病毒結構蛋白和七種病毒非結構蛋白。C型肝炎病毒感染引發慢性肝疾病,諸如肝炎、肝臟脂肪變性、肝硬化和肝癌。目前研究已建立攜帶報導 (螢光酶或綠螢光蛋白) 基因之複製子 (replicon) 或具感染性合成病毒來同步反應細胞內病毒的複製。然而報導基因直接與C型肝炎病毒基因之構築連接會減弱病毒之複製與降低感染性病毒之產量。為了解決嵌合複製子低複製能力與低病毒產量的困境,我們建立C型肝炎病毒感染報導細胞株,JFH1-SEAP細胞株,其報導基因未與C型肝炎病毒基因直接構築連接。JFH1-SEAP細胞株表現由慢病毒 (lentivirus) 置入之報導合成基因,產生綠螢光蛋白 (enhancement green fluorescence protein) - HCV NS3/4 蛋白酶之 △4AB目標肽 - 分泌性鹼性磷酸酶 (secreated alkaline phasphatase, SEAP) 之合成蛋白,同時細胞內之C型肝炎病毒基因型 2a之全長基因能複製產生具感染性C型肝炎病毒顆粒。 C型肝炎病毒基因能轉譯出具功能之HCV NS3/4 蛋白酶以切割 △4AB目標肽,將 SEAP 釋出。藉由培養液中之SEAP 活性反映細胞內HCV之複製。結果顯示, JFH1-SEAP 細胞株能表現病毒蛋白,且以複製生長的方式等差增加 SEAP活性比值,使SEAP活性比值線呈線性上升。此外我們同時培養 JFH1-SEAP 細胞株和 HuH7.5-SEAP 細胞株模擬C型肝炎病毒顆粒感染細胞,並藉JFH1-SEAP 細胞之單種細胞株或混合細胞株培養系統篩選具抗病毒特性之生化活性物質。結果顯示,干擾素在JFH1-SEAP 細胞之單種細胞株或混合細胞株培養系統中表現抗C型肝炎病毒之能力,而維生素 E能促使HCV複製。在藥物篩選實驗中,葉酸、表皮生長因子、氯化鋰和酯蛋白脂酶在藥物處理後第四天發現SEAP 活性下降。綜合實驗結果,我們建立之C型肝炎病毒持續性感染報導細胞株,JFH1-SEAP細胞株,除了能用於偵測C型肝炎病毒於細胞內之複製與感染,亦能為抗C型肝炎病毒藥物篩選有利之平臺。
英文摘要 Hepatitis C virus (HCV) contains a single-stranded positive-sense RNA genome that encodes 3 structural and 7 non-structural proteins. HCV infection may lead to chronic liver disease, including hepatitis, steatosis, cirrhosis, and hepatocellular carcinoma. Recently, HCV replicons or chimeric viruses that contained reporter genes (luciferase or green fluorescence protein) have been useful to study HCV replication in cells. However, the reporter genes inserted into HCV genome may attenuate HCV replication and subsequent virus production. To avoid the drawbacks of chimeric replicons, we established a HCV-producing cell line, JFH1-SEAP cells, with separatedly expressed reporter gene and HCV genome. The JFH1-SEAP cells expressed enhancement green fluorescence protein-NS3/4 protease-cleavable △4AB peptide-secreted alkaline phosphatase fusion protein that was transduced by lentivirus and replicating HCV genotype 2a full-length sequences to produce infectious HCVcc. The functional HCV NS3/4 protease could cleave the △4AB peptide to release SEAP for quantitative measurement of virus replication. The SEAP activity was increased linearly as JFH1-SEAP replicated. Moreover, we cultured JFH1-SEAP cells and HuH7.5-SEAP cells simultaneously to allow HCV infection to the naïve cells. Agreed with the previous reports, our results showed that interferon-α prohibited the anti-HCV activity. Vitamin E enhanced HCV replication. Furthermore, folic acid, EGF, LiCl and LPL decreased the SEAP activity. Our results demonstrated that a novel HCV-producing cell line with a SEAP reporter, JFH1-SEAP cell, was established. The system is a powerful platform to monitor HCV infection or replication and holds a potential for high-throughput investigation of the effects of nutrients and biochemical compounds on HCV replication.
論文目次 中文摘要 I
英文摘要 (Abstract) II
誌謝 III
目錄 IV
圖表目錄 IX
藥品與儀器 XII
第一章、緒論 1
I. C型肝炎病毒概論 1
1.1 C型肝炎病毒之基因結構 1
1.2 C型肝炎病毒之基因型分類及其分佈 1
1.3 C型肝炎病毒之生活史 2
1.3-1 病毒進入細胞 2
1.3-2 病毒轉錄與複製 3
1.3-3 病毒組裝與出芽 4
1.4 C型肝炎病毒之致病機轉 4
1.4-1 C型肝炎病毒和肝癌 4
1.4-2 C型肝炎病毒和脂質代謝 4
1.5 C型肝炎病毒之治療 5
1.5-1 干擾素合併 Ribavirin 治療 5
1.5-2 營養物合併治療 6
II. 目前研究 C 型肝炎病毒之實驗系統 7
2.1 細胞培養實驗系統 8
2.1-1 C 型肝炎病毒之血清感染系統 8
2.1-2 C 型肝炎病毒之基因複製子系統 8
2.1-3 C 型肝炎病毒培養系統 9
2.1-4 C 型肝炎病毒偽顆粒 10
2.2 動物培養實驗系統 10
III. 報導系統 10
IV. 實驗目標和實驗設計 14
第二章、材料與方法 15
I. 細胞株 15
1.1 HuH-7 15
1.2 cured HuH-7 15
1.3 HuH 7.5-SEAP 15
1.4 FL/J15 15
II. 細胞培養系統 16
2.1 基本培養 16
2.2 冷凍細胞 17
2.3 解凍細胞 18
III. HCVcc 之純化與定量 18
3.1 HCVcc 之純化 18
3.2 HCVcc 之定量 19
3.2-1 以未知濃度的病毒感染細胞 19
3.2-2 免疫螢光染色法 20
3.2-3 TCID50 定量病毒濃度 21
IV. 利用流式細胞儀分析病毒感染細胞之比例 22
4.1 已知濃度病毒感染細胞 22
4.2 JFH1-SEAP 細胞株與 FL/J15 細胞株之感染細胞分析 23
4.3 流式細胞儀分析 23
V. 用 LipofectamineTM2000 進行 HCV 表現質體之轉染 25
VI. JFH1-SEAP 細胞內外之 HCV RNA 定量 26
6.1 細胞內 RNA 之萃取 26
6.2細胞外 RNA 之萃取 27
6.3 RNA 之反轉錄 (reverse transcription) 28
6.4 以 real-time PCR 定量 HCV RNA 29
VII. 細胞外分泌性鹼性磷酸酶活性之分析 30
7.1預備細胞 30
7.1-1 JFH1-SEAP細胞與感染HuH7.5-SEAP細胞 30
7.1-2 混和培養不同比例之 JFH1-SEAP 細胞和 Huh7.5-SEAP 細胞31
7.2 偵測分泌性鹼性磷酸酶之活性 31
VIII. 西方點墨法分析 HCV 病毒蛋白之表現 33
8.1 細胞總蛋白質收集 33
8.2 細胞總蛋白質定量 34
8.3聚丙烯醯胺膠體電泳 34
8.4 蛋白質轉漬 (transfer) 和Blocking 36
8.5 西方點墨分析 37
IX. 油紅染色觀察細胞內之脂肪小滴 38
9.1 油紅染色法 38
9.2 油紅定量 39
第三章、結果 40
I. 建立C型肝炎病毒感染報導細胞株JFH1-SEAP 40
II. 分析JFH1-SEAP 細胞株的特性 40
2.1 JFH1-SEAP 細胞株可以產生具有效感染性的病毒 40
2.1-1 HCV RNA能在JFH1-SEAP 細胞株中複製 40
2.1-2 不同濾孔純化之病毒對細胞感染並無影響 40
2.1-3 從JFH1-SEAP 細胞株純化之病毒可以感染細胞 42
2.2 JFH1-SEAP細胞可以表現HCV病毒蛋白 42
2.2-1 HCV core、NS3與NS5A病毒蛋白表現在JFH1-SEAP細胞之細胞質 42
2.2-2 JFH1-SEAP 細胞株不均勻表現病毒蛋白 43
2.3 JFH1-SEAP細胞釋出的SEAP活性可以同步反應細胞內HCV複製情況44
2.4 JFH1-SEAP細胞以複製生長而非感染的方式增加HCV感染細胞45
2.5 JFH1-SEAP細胞內含大量脂肪小滴 46
III. JFH1-SEAP細胞株的應用 47
3.1 混合培養JFH1-SEAP細胞與Huh7.5-SEAP細胞能模擬傳統病毒感染47
3.1-1 不同比例混合培養感染細胞的SEAP活性比值以非線性曲線上升47
3.1-2 以1:49比例混合培養感染細胞的病毒擴散感染速度與病毒感染 (MOI 0.01)細胞相似 48
3.1-3 不同比例混合培養感染細胞之 Core、NS3與NS5A表現具劑量依賴性 48
3.1-4 不同比例之混合培養感染細胞均以病毒擴散的方式增加HCV (+)細胞 49
IV. 應用 JFH1-SEAP 細胞探討生化活性物質對 C 型肝炎病毒之影響50
4.1干擾素之抗 C 型肝炎病毒的複製能力 50
4.2 維生素 E 增加C 型肝炎病毒複製之能力 51
4.3 利用混合感染 (1:49 比例) 報導系統進行篩選具抗病毒特性的生化活性物質 53
4.4 酯蛋白脂酶之抗 C 型肝炎病毒的能力 54
4.5 上皮細胞生長因子影響 C 型肝炎病毒的能力 55
第四章、討論 56
I. 比較目前 C 型肝炎病毒感染系統與 JFH1-SEAP 感染報導系統應用在篩選抗 HCV 藥物之優劣 56
II. HCVcc感染報導系統可能存在的障礙 57
III. JFH1-SEAP 感染報導系統可能存在的障礙 57
3.1 JFH-1 病毒之適應性突變 (adaptive mutation) 57
3.2 EGFP-SEAP 報導蛋白表現下降 58
IV. 篩選對 HCV 有影響之物質其可能抗C 型肝炎病毒的機制 58
4.1干擾素在JFH1-SEAP單一細胞報導系統、混合感染報導系統及病毒感染報導 系統之抗C 型肝炎病毒複製的效果 58
4.2 維生素E之促C 型肝炎病毒複製的機制 59
4.3 上皮細胞生長因子之抗C 型肝炎病毒的機制 60
4.4 氯化鋰之抗C 型肝炎病毒的機制 61
4.5 葉酸之抗C 型肝炎病毒的機制 62
4.6 酯蛋白脂酶之抗 C 型肝炎病毒的機制 62
V. 結論 63
第五章、文獻 64
表 75
圖 77
附圖 119
作者簡歷 123
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