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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張明富 | zh_TW |
dc.contributor.author | 植錦炤 | zh_TW |
dc.contributor.author | Kam-Chio Chek | en |
dc.date.accessioned | 2021-06-17T03:40:38Z | - |
dc.date.available | 2023-11-30 | - |
dc.date.copyright | 2018-03-29 | - |
dc.date.issued | 2018 | - |
dc.date.submitted | 2002-01-01 | - |
dc.identifier.citation | 1. Burns, G. S., and Thompson, A. J. (2014) Viral hepatitis B: Clinical and epidemiological characteristics. Cold Spring Harbor Perspectives in Medicine 4, a024935
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70047 | - |
dc.description.abstract | B型肝炎病毒(Heptitis B virus, HBV),屬於嗜肝DNA病毒科(hepadnaviridae),感染此病毒會引發急性或慢性B型肝炎,而慢性B型肝炎又有機會進展成肝硬化與肝癌。HBV為有套膜的部分雙股DNA病毒,長度約為3.2 kbp (kilo base pairs),主要透過接觸病人的血液或體液而感染。HBV的外套膜上含有三種表面抗原蛋白質(HBsAg)的類型,分別是小型、中型和大型表面抗原蛋白質,其中大型表面抗原被認為在HBV的感染中扮演重要角色。在近年針對B型肝炎的研究指出,位於肝臟的human sodium taurocholate co-transporting polypeptide (hNTCP)為HBV與D型肝炎病毒(HDV)的受體。hNTCP為一穿膜蛋白質,在肝腸循環中參與鈉離子與膽鹽的運輸角色,而且只專一地於肝臟表現。當HBV進行感染時,其大型表面抗原上的pre-S1區域會進行肉荳蔻酰化(myristoylation),並與hNTCP進行結合。研究指出在人類肝臟細胞中表現hNTCP可以支持HBV的感染,但HBV卻無法在表現hNTCP的小鼠肝臟細胞中完成生活史,顯然於人類的細胞當中含有被感染的其他必要因子。另外,先前的研究發現,HBV是透過網格蛋白相關的內吞作用(clathrin-mediated endocytosis)進入肝臟細胞,並且與其中的輔助蛋白質AP-2有交互作用;與此同時,本實驗室的研究指出,由LMBRD1基因所編碼的LMBD1蛋白質也會與AP-2有交互作用,並透過類似機制調控胰島素受體的內化作用。為了暸解LMBD1蛋白質與HBV的相關性,本研究先以GST-pulldown assay證實LMBD1蛋白質與B型肝炎病毒大型表面抗原的pre-S1區域有交互作用,再以免疫共沉澱法證實了LMBD1蛋白質與hNTCP有交互作用。為了探討LMBD1蛋白質在HBV的生活史中所扮演的角色,本研究以HepG2.2.15細胞所產生的HBV與HuS-E/2細胞建立感染性HBV細胞培養系統(infectious HBV cell culture system,infectious HBVcc system)。首先以帶有LMBRD1 shRNA的慢病毒(lentivirus)感染HuS-E/2細胞,使其降低LMBD1蛋白質之表現,再以HBV感染LMBRD1已踢弱(knockdown)的HuS-E/2細胞。結果發現在LMBRD1踢弱的細胞中,HBV的基因體含量會減少;且在共軛焦顯微鏡成像觀察到,HBV核心蛋白質亦會減少。反之,若在被踢弱的細胞中過量表現LMBD1的重組蛋白質,可以部份挽救HBV感染所減少的現象。另一方面,在過去的文獻中指出,表皮生長因子受體(EGFR)的磷酸化會影響hNTCP的內吞作用;而本研究在LMBRD1踢弱的細胞中亦發現EGFR的磷酸化會減少,同時細胞膜上hNTCP表現量相較對照組會有明顯上升。綜合以上結果,LMBD1蛋白質可能透過調節細胞膜上EGFR的磷酸化,從而控制細胞膜上hNTCP的內吞作用,最終影響HBV的感染量。 | zh_TW |
dc.description.abstract | The hepatitis B virus (HBV), which belongs to the family of hepadnaviridae, is a major cause of human liver diseases. HBV is an enveloped virus that contains a partially double-stranded DNA of 3.2 kbp in length. The HBV envelope is comprised of 3 different forms of HBV surface antigen (HBsAg), the small (S), middle (M), and large (L) envelope proteins. The LHBsAg plays a pivotal role in HBV infection. HBV enters susceptible human hepatocytes through sodium taurocholate co-transporting polypeptide (hNTCP), an integral membrane glycoprotein known to participate in the enterohepatic circulation of bile acids and function as a sodium/bile acid co-transporter. There are two homologous transporters involved in the reabsorption of bile acids, but only hNTCP is expressed in the liver. Recently, hNTCP was demonstrated to be a functional receptor specific for HBV and hepatitis delta virus. hNTCP specifically interacts with the myristoylated pre-S1 domain of LHBsAg and mediates viral entry. Human hepatocytes expressing hNTCP could support HBV infection, but HBV life cycle is restricted in mouse hepatocytes expressing hNTCP. Thus, it is likely that additional host factors are required for full HBV infection. In addition, studies demonstrated that the pre-S1 domain of LHBsAg specifically interacted with clathrin heavy chain and its adaptor protein AP-2, suggesting that the entry of HBV into human hepatocytes is through clathrin-mediated endocytosis. Our previous results demonstrated that LMBD1, encoded by the limb region 1 (LMBR1) domain containing 1 gene (LMBRD1), interacts with AP-2 and is involved in the clathrin-dependent endocytosis of the insulin receptor. In order to investigate the relationships among LMBD1, hNTCP and HBV, GST pull-down assay was performed. The results demonstrated that LMBD1 interacted with the pre-S1 domain of LHBsAg. In addition, immunoprecipitation assay demonstrated an association of LMBD1 with hNTCP. Furthermore, an infectious HBV cell culture system was established in this study by infecting an immortalized primary hepatocytes HuS-E/2 with HBV particles released from HepG2.2.15 cells. When LMBRD1 gene was knocked down (K/D) in HuS-E/2 cells by lentiviruses expressing LMBRD1 shRNA, the copy number of HBV genome and the expression of HBV core protein were both decreased. These phenomena can be partially rescued by overexpression of LMBD1 in the K/D cells. Previous studies show that the phosphorylation of EGFR may upregulate hNTCP endocytosis. In this study, a lower level of EGFR phosphorylation in LMBRD1 K/D cells was detected, whereas a higher level of hNTCP on the cell membrane was detected. In conclusion, these results suggest that LMBRD1 K/D inhibits EGFR phosphorylation, resulting in the reduction of hNTCP endocytosis, and thus affect HBV infection. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T03:40:38Z (GMT). No. of bitstreams: 1 ntu-107-R04442034-1.pdf: 6409264 bytes, checksum: f7505b4722dfd5567479cf4ba97570f7 (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 誌謝 ................................ ................................ ................................ ................................ ... I
中文摘要 ................................ ................................ ................................ .......................... II Abstract ................................ ................................ ................................ ........................... IV 縮寫表 ................................ ................................ ................................ ............................ VI 第一章、 緒論 ................................ ................................ ................................ .......... 1 1. B型肝炎病毒 ................................ ................................ ................................ .. 1 1.1. B型肝炎病毒之發現史 ................................ ................................ ...... 1 1.2. B型肝炎病毒之感染方式與流行學 型肝炎病毒之感染方式與流行學 ................................ .............. 2 1.3. B型肝炎病毒的結構、基因體與蛋白質 型肝炎病毒的結構、基因體與蛋白質 ................................ .. 2 1.4. B型肝炎病毒生活史 ................................ ................................ .......... 4 1.5. B型肝炎病毒功能性受體 型肝炎病毒功能性受體 (Functional receptor) ............................... 5 1.6. B型肝炎病毒研究模式系統 型肝炎病毒研究模式系統 ................................ .............................. 6 2. LMBRD1基因與 LMBD1蛋白質 ................................ ................................ .. 7 2.1. LMBRD1的基因特性 ................................ ................................ .......... 7 2.2. LMBD1蛋白 質的功能探討 質的功能探討 ................................ ................................ 8 第二章、 研究主題 ................................ ................................ ................................ .. 9 第三章、 材料來源 ................................ ................................ ................................ 10 1. 藥品 ................................ ................................ ................................ ................ 10 2. 酵素 ................................ ................................ ................................ ................ 12 3. 抗體 ................................ ................................ ................................ ................ 12 3.1. 初級抗體 (Primary antibodies) ................................ ................................ . 12 3.2. 次級抗體 (Secondary antibodies) ................................ ............................. 13 4. 細胞培養液及試劑 ................................ ................................ ........................ 13 5. 細胞株 ................................ ................................ ................................ ............ 14 6. 套裝試劑 ................................ ................................ ................................ ........ 14 7. 質體 ................................ ................................ ................................ ................ 14 8. 引子 ................................ ................................ ................................ ................ 16 第四章、 實驗方法 ................................ ................................ ................................ 17 1. 細胞株繼代培養 (Subculture) ................................ ................................ ...... 17 2. GST pull-down assay與免疫共沉澱 與免疫共沉澱 (Co-immunoprecipitation) ................ 18 2.1. 勝任細胞 (Competent cells)的製備 ................................ .................. 18 2.2. 細菌轉型 (Transformation) ................................ ............................... 19 2.3. 質體之小量製備 (Mini-preparation of plasmid DNA) .................... 19 2.4. 質體之中量製備 (Midi-preparation of plasmid DNA) .................... 20 2.5. DNA轉染 (Transfection) ................................ ................................ .. 21 2.6. 細胞內蛋白質之收集 ................................ ................................ ........ 21 2.7. GST融合蛋白質之 表現與純化 ................................ ........................ 22 2.8. GST- pull down assay ................................ ................................ ......... 23 2.9. 免疫共沉澱 (Co-immunoprecipitation) ................................ ............ 23 2.10. 蛋白質定量正十二烷硫酸鈉 —聚丙烯醯胺膠體電泳 (SDS-polyacrylamide gel electrophoresis, SDS-PAGE) ................................ .......... 24 2.11. 西方墨點法 (Western blot analysis) ................................ ................. 25 2.12. Coomassie brilliant blue (CBR)染色法 ................................ ......... 26 3. 帶有 shRNA的 vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentivirus之製備與感染 ................................ ................................ ................ 27 3.1. 製備帶有 shRNA的 VSV-G pseudotyped lentivirus ........................ 27 3.2. 以帶有 shRNA的 VSV-G pseudotyped lentivirus作感染 ............... 28 4. B型肝炎病毒 之製備與感染 製備與感染 ................................ ................................ ........ 28 4.1. B型肝炎病毒顆粒之收集 型肝炎病毒顆粒之收集 ................................ ................................ 28 4.2. B型肝炎病毒之感染 ................................ ................................ ........ 29 5. 細胞內基因體 DNA與病 毒基因體 DNA之偵測 之偵測 ................................ ....... 29 5.1. 細胞內基因體 DNA之收集 之收集 ................................ ............................. 29 5.2. 聚合酶連鎖反應 (Polymerase chain reaction, PCR) ........................ 30 5.3. DNA瓊脂膠電泳 (DNA agarose gel electrophoresis) ..................... 30 5.4. 即時聚合酶鏈鎖反應 (real-time PCR) ................................ ............ 30 6. 免疫螢光染色 (Immunoflurescence staining assay) ................................ .... 31 7. 流式細胞儀檢測技術 (Flow cytometry) ................................ ...................... 32 第五章、 實驗結果 ................................ ................................ ................................ 33 1. LMBD1蛋白質、 B型肝炎病毒 型肝炎病毒 與 hNTCP之間的交互作用 .................... 33 2. 建立 HBV細胞培養系統 細胞培養系統 ................................ ................................ .............. 34 2.1. 製備並純化 HBV病毒顆粒 病毒顆粒 ................................ .............................. 34 2.2. 以 HuS-E/2細胞株建立病毒感染系統 ................................ ............ 35 3. 建立 VSV-G pseudotyped lentivirus轉導之 LMBD1基因踢弱系統與過量 表現系統 ................................ ................................ ................................ ........ 35 3.1. 細胞中 LMBRD1基因的踢弱會減少 基因的踢弱會減少 HBV的感染量 的感染量 ................... 35 3.2. 外送 LMBD1蛋白質表現體可以部份挽救因 蛋白質表現體可以部份挽救因 LMBD1踢弱所造 成 HBV感染量減少的情況 感染量減少的情況 ................................ ................................ .......... 36 4. 細胞中 LMBD1基因的 踢弱會導致細胞表面基因的 踢弱會導致細胞表面hNTCP表現量上升 ...... 37 5. LMBD1蛋白質、 EGFR與 hNTCP之間的關係 之間的關係 ................................ ........ 38 5.1. 細胞中 LMBD1基因的踢弱會使 基因的踢弱會使 EGFR的磷酸化減少 的磷酸化減少 ................ 38 5.2. 外送 LMBD1蛋白質表現體可增加 蛋白質表現體可增加 EGFR的磷酸化 的磷酸化 ................ 38 第六章、 討論 ................................ ................................ ................................ ........ 40 1. 本研究使用 HuS-E/2細胞株的優點 ................................ ............................ 41 2. LMBD1參與 hNTCP內吞作用的可能性 內吞作用的可能性 ................................ ................... 41 3. LMBD1參與在溶酶體中協助 參與在溶酶體中協助 hNTCP送出 HBV至細胞質之可能性 ..... 43 4. 結語 ................................ ................................ ................................ ................ 43 第七章、 圖表 ................................ ................................ ................................ ........ 44 第八章、 附圖 ................................ ................................ ................................ ........ 57 第九章、 參考文獻 ................................ ................................ ................................ 62 | - |
dc.language.iso | zh_TW | - |
dc.title | LMBD1蛋白質在B型肝炎病毒感染中扮演之角色 | zh_TW |
dc.title | Roles of LMBD1 protein in hepatitis B virus infection | en |
dc.type | Thesis | - |
dc.date.schoolyear | 106-1 | - |
dc.description.degree | 碩士 | - |
dc.contributor.oralexamcommittee | 劉旻禕;余明俊;董馨蓮 | zh_TW |
dc.contributor.oralexamcommittee | ;; | en |
dc.subject.keyword | B型肝炎病毒,人類NTCP,LMBD1蛋白質,表皮生長因子受體,病毒感染, | zh_TW |
dc.subject.keyword | HBV,hNTCP,LMBD1,EGFR,virus infection, | en |
dc.relation.page | 75 | - |
dc.identifier.doi | 10.6342/NTU201800197 | - |
dc.rights.note | 未授權 | - |
dc.date.accepted | 2018-02-08 | - |
dc.contributor.author-college | 醫學院 | - |
dc.contributor.author-dept | 生物化學暨分子生物學研究所 | - |
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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