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研究生:張容慎
研究生(外文):Jung-Shen Chang
論文名稱:STAT1在登革病毒感染的HepG2細胞中調控IL-8表現及其機轉
論文名稱(外文):STAT1 regulates IL-8 production in dengue virus-infected HepG2
指導教授:何令君賴振宏賴振宏引用關係
指導教授(外文):Ling-Jun HoJenn-Haung Lai
口試委員:林昌棋
口試日期:2011-06-28
學位類別:碩士
校院名稱:國防醫學院
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:66
外文關鍵詞:dengue
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登革病毒是屬於黃質病毒科(Flaviviridae)中的黃熱病毒,為正股的RNA病毒,以血清型的不同,分為第一型到第四型。登革病毒主要是藉由埃及斑蚊(Aedes aegypti)和白線斑蚊(Aedes albopictus)傳播到人類,感染登革病毒會導致病情較輕微的登革熱,或是較嚴重的登革出血熱(DHF)和登革休克症候群(DSS),致病原因是因為不正常的免疫反應使得細胞激素過度表現、T細胞的活化,並造成血液恆定失去平衡。其中趨化性細胞激素(chemokine)會增加發炎反應並和疾病的致病機轉有密切關係,當登革病毒感染巨噬細胞、內皮細胞、肝細胞、肥大細胞後,IL-8的表現量會增加。在臨床研究中發現,登革病毒感染肝細胞後會造成肝細胞損傷,導致肝脾腫大,目前對於登革病毒感染肝細胞所誘發的細胞激素而導致的致病機轉仍不清楚,所以在本實驗用HepG2細胞當實驗model。
已知登革病毒感染樹突細胞會活化STAT1和STAT3,且STAT會參與細胞激素活化的傳導路徑,因此本實驗想探討登革病毒感染HepG2細胞後,STAT的活化情形,並進一步看活化的STAT如何調控病毒所誘發的趨化性細胞激素。
實驗結果顯示,登革病毒感染 HepG2細胞24小時後,STAT1就被活化,且total STAT1的表現量在48小時也增加,STAT3則是在感染後24小時被活化起來,但是到48小時,就沒有觀察到活化的現象。knockdown STAT1後,由ELISA、Q-PCR方式可發現登革病毒所誘發的IL-8、RANTES的表現量都減少了,表示STAT1這些細胞激素的誘發過程中,扮演著重要的角色。接下來往它們的上游來看STAT1調控IL-8表現的機轉為何,已知登革熱病毒感染細胞後,NF-κB及AP-1與DNA結合活性會增加,而NF-κB及AP-1也已被報導會參與IL-8表現,由EMSA結果發現,抑制STAT1後,被病毒感染後的NF-κB與DNA結合的活性沒有改變;而AP-1與DNA結合的活性卻只有些微的被抑制,因此推論STAT1去抑制被病毒誘導產生的IL-8的表現是經由其他路徑來調控的,目前用ChIP assay的方式來看STAT1是否藉著直接結合在IL-8 promoter上而調控被病毒誘導產生的IL.8的表現。

Dengue virus (DENV) is a member of the Flavivirus genus of the Flaviviridae family of enveloped, positive-strand RNA viruses. DENV causes a wide range of diseases in humans, from the acute febrile illness dengue fever (DF) to life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The pathogenesis of dengue virus infection is thought to involve dysregulation of the immune response. Chemokines have been proposed to contribute to enhance inflammation and disease pathogenesis. Previously studies have shown that DENV infection increased IL-8 production in primary monocyte/macrophages, endothelial cells and liver cells. According to that liver dysfunction is a characteristic of severe dengue infection, we use HepG2 cell line as the cell model in our study.
We previously reported that STATs (Signal Transducer and Activator of Transcription) activated in DENV infected DC. STATs are transcriptional factors involved in cytokine signal transduction. However, the mechanisms of how STATs activation regulated the DENV mediated inflammatory response in liver still remains unclear. In this study, we aim to investigate the role of STATs activation in DENV induced IL-8 production in HepG2 cells.
Our preliminary data showed that elevated IL-8 production and activated STAT1 and STAT3 in DENV infected HepG2 cells. Knockdown STAT1 suppress IL-8 production in DENV infected HepG2. This indicated that STAT1 may play critical role in regulate IL-8 production after dengue infection. We plan to examine how the STATs regulate IL-8 production. Studies have reported that NF-kB, AP-1 and C/EBP are transcriptional factors that regulate IL-8 production. Therefore, we use Electrophoretic Mobility Shift Assays (EMSA) to investigate whether the DENV activated NF-kB, AP-1 and C/EBP DNA-binding activities are affected after STAT1 knockdown. The result showed that knockdown STAT1 not affect the NF-kB DNA binding activity and slightly suppress of the AP-1 DNA binding activity. This demonstrates that IL-8 may directly regulated by STAT1 binding. We are currently using ChIP assay to detection the STAT1 DNA binding site on IL-8 promoter.

第一章 緒論
第一節、登革病毒 (dengue virus)..............................................................1
第二節、登革病毒和IL-8的關係...............................................................2
第三節、STAT (Signal Transducers and Activators of Transcriptio)..........3
第四節、研究目的與策略...........................................................................5



第二章 材料與方法
第一節、細胞培養與製備knock down STAT1的HepG2細胞..............6
第二節、西方點墨法 (Western blotting)...................................................8
第三節、酵素結合免疫吸附法 (enzyme.linked immunosorbent
assay﹐ELISA)..........................................................................13
第四節、電泳速度變動分析法 (electrophoretic mobility shift
assay﹐EMSA).........................................................................15
第五節、溶菌斑試驗 (plaque assay).......................................................20
第六節、定量即時聚合酶鏈鎖反應(quantitative real time
polymerase chain reaction﹐Q-PCR).......................................23
第七節、染色質免疫沈澱法(ChIP assay) ..............................................27

第三章 結果
第一節、研究被登革病毒感染的HepG2細胞中STAT的
活化情形.................................................................................33
第二節、探討登革病毒感染knockdown STAT1 的HepG2
細胞後STAT1的表現情形....................................................33
第三節、STAT1對登革病毒感染所誘導的發炎相關細胞激素
的影響.....................................................................................34
第四節、研究STAT1對登革病毒感染細胞後的新病毒產生
能力.........................................................................................35
第五節、研究STAT1對細胞內轉錄因子NF-κB及AP-1與
DNA結合活性的表現............................................................36



第四章 討論......................................................................................................38

第五章 參考文獻..............................................................................................48
圖目錄
圖1A、HepG2細胞被登革病毒感染而活化的STAT....................................52
圖1B、HepG2細胞被登革病毒感染後不會活化的STAT............................53
圖2、探討登革病毒感染knockdown STAT1 的HepG2細胞後
STAT1 的表現情形................................................................................54
圖3A、STAT1抑制登革病毒所誘導的IL-8、RANTES的蛋白質表現........55
圖3B、STAT1抑制登革病毒所誘導的IL-8、RANTES的mRNA表現…..56
圖4、STAT1不會影響登革病毒的新病毒產生能力.......................................57
圖5、STAT1不會影響登革病毒感染HepG2細胞後的NF-κB及AP-1
與DNA結合的活性.................................................................................58
附錄圖目錄

圖1A、以電泳來確認DNA shearing片段大小............................................59
圖1B、經過測試後理想的DNA片段大小...................................................60
圖2、以western確認免疫沉澱作用與否......................................................61
圖3、PCR分析STAT1是否會結合在IL-8 promoter上..............................62
圖4、用已報導過的結果當ChIP assay實驗過程的control,以western確認
免疫沉澱作用與否...............................................................................63
圖5、以PCR分析STAT3是否會結合在junB promoter上………………..64
圖6、用western確認做免疫沉澱前後,蛋白質是否有lost………………..65
圖7. 用TBP(TATA binding protein)當作internal control,並用PCR分析
TBP表現……………………………………………………………...…66

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