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Title: | 以表現鈉離子-牛磺膽酸共轉運蛋白之肝癌細胞株探討B型肝炎病毒感受性之分析 Establishment of Hepatoma Cell Line with Sodium Taurocholate Co-transporting Polypeptide for Efficient HBV Infection |
Authors: | Chen-Yen Chung 鍾承諺 |
Advisor: | 陳培哲(Pei-Jer Chen) |
Keyword: | B型肝炎,鈉離子-牛磺膽酸共轉運蛋白,蛋白激?, HBV,NTCP,PKC, |
Publication Year : | 2015 |
Degree: | 碩士 |
Abstract: | 慢性B 型肝炎病毒帶原者在全球約有2.4億,這些帶原者有很高的危險因子會發展為肝癌,因此仍然是世界上尚未解決的感染性疾病。細胞感染模式的建立對於研究B肝病毒及其衛星病毒D肝病毒的生長循環、病程及新抗病毒藥的開發而言相當重要。過去二十多年來,人類及樹鼩的初代肝細胞常被使用來研究自然的B肝病毒感染模式。然而在實驗上廣泛被使用之人類肝癌細胞株像是Huh7 及HepG2並無法自然的被B肝病毒所感染。另一方面,B肝病毒在肝細胞上的功能性受器在之前的研究都沒有被找到,因此長久下來也受限了B肝病毒學的發展。在2012年由中國李文輝教授領導的研究團隊發表的文章中,他們使用了光化白胺酸修飾的短片段B肝大型表面抗原去找出樹鼩初代肝細胞上與其作用的關鍵蛋白。最後找到了鈉離子-牛磺膽酸共轉運蛋白( Sodium Taurocholate Co-transporting Polypeptide, NTCP )可能是B肝的細胞表面受器之一,並成功的使得表達人類NTCP的人類肝癌細胞被B肝及D肝病毒感染。根據這兩年的文獻我們發現,NTCP表達的肝癌細胞株雖然可以支持病毒的感染但是感染的效率比起先前之初代細胞自然感染模式差距相當大。在此篇論文中,我們建立了數株表達人類NTCP之肝癌細胞株,另外加入了日本NIID的Wakita 教授團隊所提供的G2-NTCP-C4細胞株,並且利用細胞株誘導產生的B型肝炎病毒顆粒進行細胞感染來作為我們主要實驗的模式。根據前的研究發現到B肝病毒主要會透過Clathrin及其受器蛋白控制的胞吞作用進入肝細胞,此現象與NTCP本身調節細胞表面數量的內吞作用一致。NTCP的內吞作用已知是受到細胞內蛋白激酶所調控,在細胞模式中加入蛋白激酶的刺激劑 PMA 可以在短時間觀察到NTCP內吞現象的發生。因此我們進而以G2-NTCP-C4細胞株來探討在B型肝病毒接種的期間使用蛋白激酶刺激劑PMA是否會提升感染的效率。我們初步的結果發現,在G2-NTCP-C4細胞中接種B型肝炎病毒處理PMA的實驗裡,接種病毒前處理的組別於感染後5至7天在細胞培養液中偵測到相對高量的B肝病毒表面抗原以及e抗原,然而感染後第9天與控制組並無明顯提升。此外,接種HBV後24小時再進行PMA處理的實驗中,以高病毒量感染細胞的組別在感染後第9天釋放出高量的表面抗原及e抗原。從western blot以及免疫螢光的結果顯示,在感染後第9天的細胞內前處理以及後處理PMA的組別表現出較高量的HBV核心抗原蛋白。 Infection with Hepatitis B virus (HBV) is still a major health problem worldwide . Approximately 240 million people chronically infected with HBV have a greater risk of developing hepatocellular carcinoma(HCC). Cell culture systems are required to study the replication cycle of HBV and HDV infection, pathogenesis and the mechanisms of antiviral drugs. From two decades study, primary human (PHH) and tupaia hepatocytes(PTH) has been used to study HBV natural infection. Yan et al. 1 in 2012 year used HBV Large S protein-derived photo-leucine modified peptide to identify sodium taurocholate co-transporting polypeptide as a functional HBV and HDV entry receptor. Expression of full length of human NTCP in HepG2 and Huh-7 cells renders these cells susceptible to HBV and HDV2. However, recent studys demonstrated that HBV entry efficiency in these NTCP-reconstituted cells , compared to natural infection still unffective. In this study, we based on previous reports indicating that HBV and NTCP internalization were both regulated by clathrin-mediated endocytosis. First of all, we treated the cells with protein kinase C alpha activator, phorbol-ester (PMA) to do activate PKC and make NTCP translocation before and after HBV inoculation with HBV inoculum. Our data show that, pretreatment of PMA in NTCP-expressing cells had higher HBsAg , and HBsAg level in supernatant during 5~7 day post infection (d.p.i), but rebound to control level at 9 d.p.i. In contrast to pretreatment group, treating PMA after 24 hour inoculation, high multiplicity of infection (M.O.I) of inoculums group had significance increase HBsAg and HBeAg level at 9 d.p.i.. From the western blot and immunofluorescence data showed that, both PMA-treated groups had higher intracellular HBcAg expression. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/53646 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 微生物學科所 |
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