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研究生:林旻靜
研究生(外文):Min-ching Lin
論文名稱:幫助丙型肝炎病毒複製的細胞因子之研究
論文名稱(外文):Study on cellular factors facilitating HCV replication
指導教授:羅時燕
指導教授(外文):Shih-Yen Lo
學位類別:碩士
校院名稱:慈濟大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:76
中文關鍵詞:丙型肝炎
外文關鍵詞:HCV
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根據統計,全球感染丙型肝炎病毒 ( Hepatitis C virus ; HCV )的人口約有一億七千萬,感染HCV的成人有80%會導致慢性感染,造成肝臟產生纖維化、肝硬化,甚至導致肝癌。HCV屬於黃熱病毒科 ( Flaviviridae )中的肝炎病毒屬(hepacivirus)。它是具有套膜的病毒,屬於正鏈RNA,約有9.6kb的單股RNA病毒。HCV可以分成六種不同的基因型、超過100多種亞型。目前尚未有安全且有效的疫苗來預防HCV的感染。目前唯一有效地治療方式為干擾素α和 ribavirin 的合併治療,而此治療方式( 長效型干擾素α加上ribavirin ) 也只對約50%的病人是有效的,但通常也會伴隨著嚴重的副作用。

環胞黴素A ( Cyclosporin A ; CsA ) 是一種免疫抑制劑,廣泛地使用在肝臟移植的患者身上,但已有文獻發表Cyclosporin A可以有效抑制HCV的複製,Cyclosporin A藥物作用原理是去阻斷細胞內的結合蛋白親環素 ( cyclophilins ;CyP)。之後研究又發現Cyclosporin A的細胞標的是cyclophilin B,cyclophilin B能調節HCV RNA polymerase NS5B的功能,對於病毒基因體複製是有幫助的。因此,細胞因子會影響HCV的複製,並且可以做為抗病毒藥物的標的。

這個研究,欲尋找可以幫助HCV複製的細胞因子。我們利用微陣列分析法(Microarray)去分析HCV replicon細胞株和Re*細胞株之間的基因表現差異。之後,再利用shRNA將在HCV replicon細胞株中過量表現的基因各別進行降解(knock-down)來觀察此基因是否會影響HCV的複製。HCV的複製是藉由HCV NS5A蛋白表現量為指標。

其中, PLCG2、ELOVL4 和PLA1A這三種細胞因子被篩選出來,都是與脂肪代謝相關的基因。此外,我們使用PLCG2抑制劑(U73122) 和PLA1 抑制劑,也會抑制HCV的複製。其後的研究更發現,是這三種細胞因子所參與的產物會幫助HCV複製,因為在同個途徑的其他基因,如被降解,也會抑制HCV的複製。未來,我們將利用JFH-1感染系統中以及活體外肝臟組織切片的系統來證實這個結果。
Hepatitis C Virus (HCV) infects 170 million individuals around the world. Infection with HCV frequently causes chronic hepatitis and progresses to liver cirrhosis and even hepatocellular carcinoma (HCC). HCV belongs to the genus Hepacivirus, of the family Flaviviridae. HCV is an enveloped virus. Its genome is a single-stranded (9.6 kb) RNA. HCV isolates are further classified into six genotypes (1–6) and over 100 subtypes. Presently, no safe and effective vaccine is available to prevent HCV infection. Conventional treatment, using the combination of pegylated interferon-α (PEG-IFN-α) and ribavirin is only effective in about 50% of the patients and is associated with important side-effects.

Cyclosporin A ( CsA ) , an immunosuppressive agent widely used in the management of liver transplant recipients, has been reported to suppress the hepatitis C virus replication. Suppression of HCV replication by cyclosporin A is mediated by blockade of its cellular targets, cyclophilins. Cyclophilin (CyP) B, a cellular target of CsA, regulates the function of HCV RNA polymerase NS5B. Therefore, cellular factors could affect hepatitis C virus replication and could be the anti-viral targets.

In this study, we are going to search for the cellular factors that can facilitate HCV replication. Microarray analysis was used to screen for the genes differentially expressed between HCV replicon cell line and the cell line without HCV replicon(Re*). We them individually knocked-down selected genes over-expressed in HCV replicon by shRNA technology and determined their effects on the HCV replication by measuring the expression of HCV NS5A protein.

PLCG2、ELOVL4 and PLA1A were identified as candidate HCV replication facilitators through this screening system and all of these genes are involved in lipid metabolic pathways. Moreover, PLCG2 inhibitor U73122 and PLA1A inhibitor could also inhibit HCV replication. The other genes involved in the metabolic pathways, which these three genes were participated in, were also analyzed. Knock-down of these selected genes involved in the metabolic pathways could also repress HCV replication. Therefore, the end-products rather than the proteins participate in these metabolic pathways could facilitate HCV replication. We are going to confirm these results in the system using JFH-1 infectious clone and the ex vivo system using liver biopsy samples.
目錄
序論............................................................................1
一、丙型肝炎病毒................................................................1
1、丙型肝炎病毒之流行病學.......................................................1
2、丙型肝炎病毒之臨床症狀.......................................................1
3、丙型肝炎病毒之基因體結構和功能...............................................2
3-1丙型肝炎病毒之基因體結構.....................................................2
3-2丙型肝炎病毒基因體功能之結構蛋白-Core 和E1、E2............................. 3
3-3丙型肝炎病毒基因體功能之非結構蛋白-p7、NS2、NS3、NS4A、NS 4B、NS5A、NS 5B...4
4、丙型肝炎病毒之生活史.........................................................5
4-1 病毒與宿主細胞的結合........................................................5
4-2內在化(internalization) 和脫殼(uncoating)....................................5
4-3 IRES轉譯和病毒蛋白的切割(polyprotein processing)............................5
4-4 RNA 複製....................................................................6
4-5病毒組裝和釋出...............................................................6
二、丙型肝炎病毒之藥物治療......................................................7
三、丙型肝炎病毒細胞培養系統....................................................8
四、丙型肝炎病毒和脂質的關係....................................................9
五、PLA1A ( Homo sapiens phospholipase A1 member A)............................12
六、PLCG2 (Homo sapiens phospholipase C, gamma 2)..............................12
七、ELOVL4 (Homo sapiens elongation of very long chain fatty acids -like 4)....13
八、研究目標...................................................................13
實驗方法與材料.................................................................15
一、實驗方法...................................................................15
細胞培養.......................................................................15
微陣列分析法(Microarray).......................................................15
Lentiviral Vector系統..........................................................15
病毒製備(Lentivirus production)................................................16
病毒感染(Lentiviral infection).................................................17
RNA萃取........................................................................17
反轉錄聚合�○s鎖反應(RT-PCR)...................................................18
即時定量聚合�○s鎖反應(Real Time -PCR).........................................19
西方點墨法(Western blot).......................................................19
Enhanced chemiluminescemce(ECL)呈色............................................20
Alkaline phosphatase(AP)呈色...................................................21
細胞之藥物處理.................................................................22
細胞存活率測試WST1.............................................................22
二、實驗材料...................................................................24
試劑...........................................................................24
藥品...........................................................................25
實驗流程.......................................................................27
實驗結果.......................................................................28
Part 1:比較HCV replicon 與 Re*細胞株之間mRNA差異性。..........................28
Part 2:利用RNAi技術尋找幫助丙型肝炎病毒複製的細胞因子。.......................29
Part 3:利用Q-PCR和Western blot確認RNAi進行Knock-down後的結果。................30
Part 4:利用PLCG、PLA1的抑制劑抑制丙型肝炎病毒的複製,並測試細胞存活率。.......31
Part 5:PLCG或PLA1的抑制劑分別與Cyclosporin A (CsA)或Interferon α(IFN)一
起抑制丙型肝炎病毒的複製的效果。...............................................33
Part 6:利用RNAi將PLA2G7進行Knock-down後抑制丙型肝炎病毒的複製的結果。.........35
Part 7:尋找PLCG2、ELOVL4、PLA1A這三個基因參與的路徑,並利用RNAi技術去抑制
個別代謝路徑的上下游。............................................36
Part 8:利用Western blot確認RNAi進行Knock-down後的結果。.......................37
結果圖.........................................................................38
Part 1.........................................................................38
Part 2.........................................................................39
Part 3.........................................................................42
Part 4.........................................................................44
Part 5.........................................................................48
Part 6.........................................................................51
Part 7.........................................................................52
Part 8.........................................................................62
結果表.........................................................................63
Part 8.........................................................................63
討論...........................................................................64
附錄...........................................................................66
參考文獻.......................................................................72
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