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系統識別號 U0026-0708201317370900
論文名稱(中文) 探討登革病毒複製機轉:蛋白酶在登革病毒複製所扮演之角色
論文名稱(英文) Study on the mechanism of dengue virus replication: the role of proteases during dengue virus replication
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 101
學期 2
出版年 102
研究生(中文) 邱琦
研究生(英文) Chi Chiu
學號 T36001081
學位類別 碩士
語文別 中文
論文頁數 102頁
口試委員 指導教授-葉才明
口試委員-王淑鶯
口試委員-林振文
中文關鍵字 登革病毒  蛋白酶  細胞核轉錄因子 
英文關鍵字 Dengue virus  protease  NF-κB 
學科別分類
中文摘要 登革病毒感染引起的症狀有輕微的登革熱、可能致死的登革出血熱以及登革休克症候群,截至目前為止,臨床上仍然沒有發展出可以有效抑制登革病毒複製的疫苗或是藥物,其中最主要的原因為缺乏一個適合用來篩選抗病毒藥物的平台,因此,我們構築了以分泌性鹼性去磷酸酶 (secreted alkaline phosphatase, SEAP) 結合登革病毒蛋白酶NS2B-NS3的報導系統,連接上pEGFP-C1載體之後,利用暫時性轉染的方式表現在HuH7和HT29細胞中,接著以觀察綠色螢光蛋白 (EGFP) 的方式來推測pEG(Δ2B3)SEAP在細胞內的表現情形,從結果可以得知EGFP的表現量會隨著細胞代數的增加而有下降的趨勢,由於之前有研究顯示在埃及斑蚊的腸道中,蛋白酶的活性會影響登革病毒的感染能力,因此我們認為宿主細胞表現的蛋白酶可能會對病毒的多蛋白進行切割,於是利用蛋白酶抑制劑來觀察蛋白酶在登革病毒複製中的作用,從結果可以得知胰蛋白酶抑制劑 (TI) 和胰凝乳蛋白酶抑制劑 (TPCK) 可以抑制登革病毒多蛋白NS2B-NS3被切割的情形,由於TPCK除了能夠抑制胰凝乳蛋白酶之外,同時也可以抑制細胞核轉錄因子 (nuclear factor kappa B, NF-κB) 的活化,而有研究顯示NF-κB活化參與了登革病毒感染引起的細胞自噬及細胞凋亡,所以我們接著利用其他NF-κB抑制劑進行實驗,由於文獻指出NF-κB活化與引起細胞自噬和細胞凋亡有關,而細胞自噬會使得登革病毒複製量上升,同時登革病毒感染會引發許多不同種類細胞的凋亡,這些現象在NF-κB被抑制之後都有顯著下降的情形,而且抑制NF-κB也可以抑制細胞激素-巨噬細胞移動抑制因子 (macrophage migration inhibitory factor, MIF) 的生成,使得發炎反應不會加劇。總結來說,我們發現宿主細胞胰蛋白酶會幫助登革病毒複製,同時NF-κB活化也會參與登革病毒感染引起的細胞自噬、細胞凋亡以及發炎激素產生,因此未來或許可以在臨床上合併使用胰蛋白酶以及NF-κB抑制劑治療受到登革病毒感染的患者。
英文摘要 Dengue virus (DENV) infection can causes dengue fever and potentially lethal diseases such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Until now, there is no effective vaccine or therapeutic drugs that can be used specifically to block DENV replication in clinic. One of the difficulties of developing anti-DENV drugs is lacking of a suitable platform for compound selection. Thus, we constructed a sequence which contains secreted alkaline phosphatase (SEAP) and dengue NS2B-NS3 cutting site, followed by ligation with vector pEGFP-C1. After transient transfection of pEG(Δ2B3)SEAP into HuH7 and HT29 cells, results showed that the EGFP signals were degraded after few passages of cell culture. According to previous studies, the midgut trypsin activity can affect DENV-2 infection and dissemination in Aedes aegypti. In order to investigate the role of proteases in DENV replication, we used inhibitors that can inhibit trypsin, chymotrypsin or both of them to understand the effect of cellular enzymes on DENV replication. Supplementation of pEG(Δ2B3)SEAP transfected cells with trypsin inhibitor revealed that the dregradation of EGFP signals was inhibited. Furthermore, tosyl phenylalanyl chloromethyl keton (TPCK), which is a chymotrypsin-like protease and NF-κB inhibitor, demonstrated the most significant inhibitory ability. We then used several NF-κB inhibitors to investigate the role of NF-κB during DENV infection. The results revealed that NF-κB not only inhibit DENV-induced autoaphgy, which is acquired for DENV replication, but also decrease the level of DENV-induced cell apoptosis, and the production of macrophage migration inhibitory factor (MIF) were also decreased after inhibition of NF-κB. Taken together, these results suggested that both trypsin-like enzymes and NF-κB activation play important roles in DENV replication, DENV-induced autophagy and apoptosis, and MIF secretion. Combination of protease and NFκB inhibitors may provide an alternative therapeutic strategy for dengue patients.
論文目次 中文摘要 I
Abstract III
致謝 V
總目錄 VI
圖目錄 XI
縮寫指引 XIII
1. 緒論 1
1.1 登革病毒 1
1.1.1 登革病毒概論與現況 1
1.1.2 登革病毒結構和非結構蛋白質 1
1.1.3 登革病毒生活史 5
1.1.4 登革病毒感染引起的病症 7
1.1.5 登革病毒與細胞自噬 8
1.1.6 登革病毒的致病機轉 9
1.2 蛋白酶在登革病毒複製過程中扮演之角色 12
1.2.1 登革病毒蛋白酶 12
1.2.2 宿主細胞蛋白酶 13
1.3 細胞核轉錄因子在登革病毒致病機轉中所扮演之角色 14
1.3.1 細胞核轉錄因子與細胞自噬 14
1.3.2 細胞核轉錄因子與細胞凋亡 15
1.4 抗登革病毒藥物的發展 16
2. 實驗動機與目標 18
3. 實驗材料與方法 19
3.1 細胞株 19
3.1.1 細胞繼代培養步驟 19
3.1.2 細胞保存 19
3.1.3 細胞解凍 20
3.2 登革病毒之製備 20
3.2.1 登革病毒來源 20
3.2.2 培養病毒之細胞株 20
3.2.3 培養登革病毒 20
3.2.4 定量登革病毒效價 21
3.3 構築登革病毒蛋白酶Δ(NS2B-NS3) 及分泌性鹼性去磷酸酶(SEAP) 之重組質體DNA 22
3.3.1 萃取登革病毒RNA 22
3.3.2 反轉錄聚合酵素連鎖反應 (RT-PCR) 23
3.3.3 聚合酵素連鎖反應 (PCR) 24
3.3.4 製備勝任細胞 (competent cell) 26
3.3.5 質體構築 (construction) 及轉型 (transformation) 27
3.4 表現登革病毒蛋白質Δ(NS2B-NS3) 及分泌性鹼性去磷酸酶 (SEAP) 之重組質體DNA 30
3.4.1 SDS-PAGE分析 30
3.4.2 西方墨點法分析 31
3.4.3 比色法分析轉染重組質體細胞釋放分泌性鹼性去磷酸酶 32
3.5 分析哺乳類動物細胞所表現之蛋白酶 33
3.5.1 萃取細胞RNA 33
3.5.2 反轉錄聚合酵素連鎖反應 (RT-PCR) 33
3.5.3 聚合酵素連鎖反應 (PCR) 34
3.6 蛋白酶及細胞核轉錄因子抑制劑 36
3.6.1 蛋白酶抑制劑 36
3.6.2 細胞核轉錄因子抑制劑 37
3.7 分析蛋白酶及細胞核轉錄因子抑制劑對登革病毒之影響 37
3.7.1 利用流式細胞儀分析登革病毒感染力 37
3.7.2 利用Fluorescence Focusing Assay (FFA) 分析登革病毒感染力 39
3.7.3 利用表現LC3-EGFP之細胞分析登革病毒引起細胞自噬 (autophagy) 之能力 40
3.7.4 利用免疫螢光分析法 (immunofluorescence assay, IFA) 分析細胞核轉錄因子抑制劑影響細胞核轉錄因子活化之能力 41
3.7.5 偵測細胞凋亡程度 41
3.7.6 利用酵素連結免疫吸附分析法 (Enzyme-Linked Immunosorbent Assay, ELISA) 分析細胞釋放細胞激素之能力 43
3.8 統計分析 45
4. 結果 46
4.1 建立以登革病毒蛋白酶為標的的抗病毒藥物篩選平台 46
4.1.1 構築登革病毒蛋白酶Δ(NS2B-NS3) junction site及分泌性鹼性去磷酸酶(SEAP) 之重組質體DNA 46
4.1.2 細胞內蛋白酶對Δ(NS2B-NS3)-SEAP表現之影響 46
4.2 細胞內蛋白酶對登革病毒複製之影響 47
4.2.1 細胞內表現蛋白酶之情形 47
4.2.2 蛋白酶抑制劑對登革病毒蛋白質被切割之影響 48
4.2.3 蛋白酶抑制劑對登革病毒複製之影響 49
4.3 細胞核轉錄因子抑制劑對登革病毒感染之影響 50
4.3.1 細胞核轉錄因子對登革病毒引起細胞自噬之影響 50
4.3.2 細胞核轉錄因子對登革病毒引起細胞凋亡之影響 51
4.3.3 細胞核轉錄因子對宿主細胞分泌細胞激素之影響 52
5. 討論 54
5.1 蛋白酶對登革病毒蛋白質之作用 54
5.2 登革病毒感染引起細胞自噬及細胞凋亡之機制 56
5.3 登革病毒感染引起細胞核轉錄因子活化對病毒致病機轉之影響 58
6. 結論 61
7. 引用文獻 62
8. 圖附錄 73
附錄一、NS2B-NS3-SEAP報導系統之機制 93
附錄二、 宿主細胞蛋白酶與細胞核轉錄因子參與登革病毒複製示意圖 94
附錄三、 實驗耗材、試劑及抗體 95
附錄四、 儀器 101
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