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系統識別號 U0026-0812200915060063
論文名稱(中文) CD14在砂眼披衣菌感染所扮演的角色
論文名稱(英文) The role of CD14 in Chlamydia trachomatis infection
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 97
學期 1
出版年 98
研究生(中文) 洪盟惠
研究生(英文) Meng-Hui Hung
學號 t3695111
學位類別 碩士
語文別 中文
論文頁數 64頁
口試委員 口試委員-邢福柳
口試委員-凌 斌
口試委員-葉才明
指導教授-林尊湄
中文關鍵字 感染力  砂眼披衣菌  CD14  NF-κB  熱休克蛋白六十 
英文關鍵字 cHSP60  NF-κB  infectivity  CD14  Chlamydia trachomatis 
學科別分類
中文摘要 砂眼披衣菌的感染可導致多種慢性發炎症性疾病。因為砂眼披衣菌含有兩種成份:脂多醣體和熱休克蛋白六十,會與藉由glycosylphosphatidylinostitol鑲崁在細胞膜表面的CD14蛋白作用,進而活化巨噬細胞引起發炎反應,是目前認為披衣菌感染造成慢性發炎病變的主要致病機轉。從我們實驗室先前的研究顯示出CD14基因啟動子的多型性(-260C>T)會影響單核球表面CD14的表現量,及個體在接受披衣菌刺激後TNF-α的產生量。因此,本研究主要的目的是想探討CD14在砂眼披衣菌衣進行感染和引發發炎反應中所扮演的角色。由實驗數據顯示,砂眼披衣菌在過度表現CD14的CHO細胞株的感染力有明顯增加,而在含有anti-CD14抗體的存在下,則會明顯減低砂眼披衣菌細胞的感染力;經由進一步的實驗發現,砂眼披衣菌侵入CHO/CD14能力遠高於沒有CD14表現的CHO/pCR3細胞,另外,利用PMA刺激U937細胞表面CD14的表現量,也證實砂眼披衣菌侵入能力與U937細胞CD14表現量有關。另外,從我們的結果也發現砂眼披衣菌的感染到後期,會促進NF-κB reporter的活性表現並與披衣菌的感染劑量呈現相關性;且砂眼披衣菌感染所誘導NF-κB reporter的活性增加的現象,在TLR2和CD14同時表現的細胞株中更加上升。最後,我們也證明在砂眼披衣菌感染的過程中,主要會經由熱休克蛋白六十而不是由脂多醣體刺激CD14而活化NF-κB。總而言之,CD14在砂眼披衣菌侵入細胞增加感染能力的過程扮演重要的角色,而砂眼披衣菌感染的過程中,生長成熟後所製造的熱休克蛋白六十是經由CD14活化NF -κB的最主要因子。
英文摘要 Infection with the Chlamydia trachomatis can lead to a variety of chronic inflammatory diseases. Two components, Chlamydial lipopolysaccharide (LPS) and heat shock protein 60 (cHSP60), that can bind to CD14, a glycosylphosphatidyl-inositol-anchored membrane protein, and activate macrophages to induce inflammatory responses, which have been shown implicated in the pathogenesis of chronic inflammatory chlamydial diseases. Our previous studies have shown that the CD14 functional gene polymorphism -260C>T is associated with CD14 expression and Chlamydia-stimulated TNF-α production. Thus, the purpose of this study is to investigate the specific roles of CD14 in C trachomatis infection and inflammatory responses. The results demonstrated that infectivity of C trachomatis is significantly higher in CD14 over-expressed CHO (CHO/CD14) cell lines. Blockade of surface CD14 using anti-CD14 could reduce the C trachomatis infectivity. We found the internalization activity of C trachomatis is higher in CHO/CD14 than CHO/pCR3 cells, and the results were similar in PMA-treated U937 cells. In addition, Chlamydia infection dose-dependently increased NF-κB reporter activity in CHO/CD14 cells compared with CHO/pCR3. The effect of CD14 on NF-κB activation was enhanced by TLR2 cotransfection. Furthermore, we have demonstrated Chlamydial HSP60 but not LPS to enhance NF-κB transactivation by CD14-dependent manner after C trachomatis infection. In conclusion, CD14 plays an important role in C trachomatis internalization to enhance infectivity. The HSP60 which is produced by Chlamydia proliferation, is the most important component to induce CD14-mediated NF-κB activation during C trachomatis infection
論文目次 中文摘要 I
Abstract II
誌謝 III
目錄 IV
表目錄 VII
圖目錄 VII
附錄 VII
縮寫檢索表 VIII
儀器及藥品 IX
儀器 IX
藥品 X
1. 緒論 1
1.1 披衣菌 1
1.1.1 披衣菌簡介 1
1.1.2 披衣菌的發育循環史 2
1.1.3 砂眼披衣菌所引發的疾病及相關機制 3
1.2 CD14 4
1.2.1 CD14和其訊息傳導 4
1.2.2 CD14與砂眼披衣菌感染的關係 5
1.2.3 CD14基因多型性與砂眼披衣菌感染的關係 6
1.3 研究目的 7
2. 研究材料與方法 8
2.1 細胞培養 8
2.1.1 繼代細胞培養 8
2.1.2 建立CHO/pCR3、CHO/CD14穩定轉染細胞株 9
2.1.3 細胞冷凍保存 11
2.1.4 細胞解凍 11
2.2 砂眼披衣菌 12
2.2.1 高效價披衣菌的培養 12
2.3 砂眼披衣菌效價測定 14
2.3.1 披衣菌感染 14
2.3.2 披衣菌包涵體的免疫螢光染色 14
2.4 CD14對砂眼披衣菌吸附與分析的影響 16
2.4.1 螢光標記砂眼披衣菌 16
2.4.2 砂眼披衣菌吸附與分析 16
2.4.3 人類單核球吞噬FITC標示的砂眼披衣菌的能力 17
2.5 NF-κB報導質體活性分析 18
2.5.1 小量質體抽取 18
2.5.2 大量質體抽取 19
2.5.3 質體的轉染 21
2.5.4 CD14 blocking assay 22
2.6 探討砂眼披衣菌活化NF-κB的影響因子 23
2.7 Recombinant chlamydial HSP60純化及表現 24
2.7.1 聚合酶連鎖反應 24
2.7.2 質體的接合反應 (Ligation) 25
2.7.3 大腸桿菌之形質轉移 (Transformation) 26
2.7.4 IPTG 誘導recombinant cHSP60 蛋白質的表現 27
2.7.5 純化recombinant cHSP60 蛋白質 28
2.8 以西方墨點法及SDS-PAGE分析GST-cHSP60蛋白 29
2.8.1 SDS-PAGE分析 29
2.8.2 西方墨點法 31
2.9 探討GST-cHSP60蛋白在CD14存在時,對於NF-κB活化的影響 33
3. 結果 34
3.1 建立CHO/ CD14和CHO/pCR3穩定轉染細胞株 34
3.2 CD14對砂眼披衣菌感染力之影響 34
3.3 CD14對砂眼披衣菌黏附及內在化的影響 35
3.4 人類單核球CD14表現對砂眼披衣菌內在化的影響 35
3.5 CD14對砂眼披衣菌活化NF-κB的影響 36
3.6 TLR2,TLR4 and CD14對砂眼披衣菌活化NF-κB的影響 37
3.7 探討影響砂眼披衣菌經由CD14活化NF-κB的因子 37
3.8 表現及純化recombinant cHSP60蛋白 38
3.9 Chlamydial HSP60在CD14存在對NF-κB活化的影響 38
4. 討論 40
5. 參考文獻 45
6. 附錄 60
7. 自述 64
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