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系統識別號 U0026-0812200911165672
論文名稱(中文) 鏈球菌致熱原性外毒素 B 引發細胞IL-8分泌及其分子機制之探討
論文名稱(英文) IL-8 production induced by streptococcal pyrogenic exotoxin B and its molecular mechanism
校院名稱 成功大學
系所名稱(中) 生物化學研究所
系所名稱(英) Department of Biochemistry
學年度 92
學期 2
出版年 93
研究生(中文) 吳祥玉
研究生(英文) Hsiang-Yu Wu
電子信箱 b8721032@student.nsysu.edu.tw
學號 s1691410
學位類別 碩士
語文別 中文
論文頁數 89頁
口試委員 口試委員-林以行
指導教授-林銘德
口試委員-莊偉哲
中文關鍵字 鏈球菌致熱原性外毒素 B  鏈球菌  細胞激素  上皮細胞 
英文關鍵字 SPE B  Group A streptococcus  cytokine  IL-8 
學科別分類
中文摘要   過去的研究指出SPE B是A群鏈球菌的重要致病因子,在鏈球菌的感染上扮演相當重要的角色。就功能上而言,SPE B是屬於一種cysteine protease,它廣泛的存在A群鏈球菌當中。細胞激素(cytokine)的分泌是人體相當重要的免疫反應,為了釐清SPE B在分泌細胞激素方面所扮演的角色,因此我們研究SPE B在A549細胞所引發的發炎相關細胞激素(proinflammatory cytokines)的分泌。
利用ELISA試驗的方式,我們發現SPE B會引發A549細胞IL-8和少量IL-6的分泌。進一步以RT-PCR的方式確認SPE B會引發A549細胞IL-8基因的表現;且利用抑制劑的實驗證明其是透過ERK pathway和轉錄因子NF-κB所引發的。

  由本實驗室之前關於SPE B所引發細胞凋亡的研究結果顯示SPE B本身的蛋白脢活性及RGD motif會影響SPE B和宿主細胞之間的交互作用。為了要了解SPE B本身的蛋白脢活性及RGD motif是否也會影響宿主細胞IL-8的分泌。因此我們純化了SPE B不具有蛋白脢活性的C192S突變蛋白和不具有RGD motif的G308S突變蛋白。實驗結果發現不具有蛋白脢活性的C192S無法引發IL-8的分泌但G308S則會。因此我們認為SPE B所引發A549細胞IL-8的分泌,蛋白脢活性扮演相當重要的角色而RGD motif並沒有參與其間的作用。由於PAR 2的agonist 在A549細胞可以引發較多IL-8的產生,我們也試著以PAR 2的抗體去抑制SPE B所引發的IL-8產生,結果無效。我們也用了其它接受器如Fas、TLR 2和TLR 4的抗體看是否可以抑制SPE B所引發的IL-8產生結果都無效。
英文摘要   Streptococcal Pyrogenic Exotoxin B (SPE B), a highly conserved cysteine protease expressed by Streptococcus pyrogenes, has been shown to be an important virulence factor in streptococcus infection. Cytokine release is one of critical immunoresponses in bacterial infection. In order to elucidate the role of SPE B in cytokine production, we investigated the IL-8 production induced by rSPE B in A549 cells.

  In this study, we found that rSPE B caused IL-8 and IL-6 production in A549 cells, as measured by ELISA assay. The expression of IL-8 mRNA, measured by RT-PCR, increased one hour after the exposure to rSPE B. Pharmacological inhibitors were used to identify the signaling pathway leading to IL-8 gene expression induced by rSPE B. Inhibition of ERK and NF-κB attenuated rSPE B-induced IL-8 production.

  Previous studies from our laboratory on the SPE B-induced apoptosis showed that both protease activity and RGD motif of SPE B were involved in the interaction of SPE B with host cells. For this study, in addition to SPE B, we have also prepared the C192S and G308S to study their role in cytokine production. The rSPE B was expressed as a 42 kDa zymogen and converted to 28kDa protease active form, the C192S mutant was expressed as 42 kDa form without protease activity. The G308S mutant is a mutant on RGD motif with protease activity. Our results showed that both 42 kDa C192S and 28 kDa C192S could not induce IL-8 production in A549 cells, and E64 (cysteine protease inhibitor) could block the rSPE B-induced IL-8 production. On the other hand, the G308S mutant could induce IL-8 production in A549 cells. These results suggest rSPE B-induced IL-8 production is protease activity dependent and RGD motif independent. Agonists of PARs were also tested on A549 cells for their effects on the production of IL-8. Agonist of PAR 2 induced more production of IL-8. However, the antibody of PAR 2 did not block the SPE B-induced IL-8 production. Antibodies of other possible receptors, such as Fas, TLR 2 and TLR 4, were also evaluated. They all could not block the SPE B-induced IL-8 production.
論文目次 中文摘要 ………………………………………………………… 1
英文摘要 …………………………………………………… 2
誌謝 ……………………………………………………… 4
目錄 ............................................. 5
圖目錄 ........................................... 8

Ⅰ緒論 ………………………………………………………. 9
Ⅱ材料與方法 ………………………………………………. 15

2.1藥劑 ………………………………………………………….. 15
2.2儀器 ………………………………………………………….. 17
2.3 ProSPE B及其突變株重組蛋白的純化 …………………… 19
2.3.1重組蛋白表現 …………………………………………………… 19
2.3.2重組蛋白之純化 ………………………………………………… 20
2.3.3 SDS-PAGE膠體製作 ……………………………………………. 21
2.3.4 SDS-PAGE膠體電泳分析 ………………………………………. 22
2.3.5重組蛋白的濃縮 …………………………………………………. 23
2.3.6去除蛋白質溶液所含LPS ……………………………………….. 23
2.3.7溶液LPS含量測試--鱟的變形血球抽出液試驗 ………….……. 24
2.3.8蛋白質定量 ………………………………………………….…… 24
2.3.9蛋白脢活性分析 ……………………………………………….. 25
2.4 28 kDa C192S的製備 ……………………………………….. 25
2.5人類呼吸道上皮細胞(A549)的培養方法 …………………… 26
2.5.1配製DMEM培養液 ……………………………………………... 26
2.5.2細胞的繼代培養 ………………………………………………….. 26
2.5.3凍細胞的方法 …………………………………………………….. 27
2.5.4解凍細胞的方法 ………………………………………………….. 28
2.5.5細胞的計數 ……………………………………………………….. 28
2.6人類單核球細胞(U-937)的培養方法 …………………………. 28
2.6.1配製RPMI-1640培養液 ………………………………………….. 29
2.6.2細胞的繼代培養 …………………………………………………… 29
2.6.3凍細胞的方法 ……………………………………………………… 30
2.6.4解凍細胞的方法 …………………………………………………… 30
2.7活化人類單核球細胞株(U-937) ………………………………... 31
2.8分離人類單核球細胞/巨嗜細胞及淋巴球細胞 ……………….. 31
2.9酵素連結免疫吸附分析(ELISA)測定細胞素(cytokine)含量 ... 33
2.9.1收集樣品 ……………………………………………………………. 33
2.9.2酵素連結免疫吸附分析(ELISA) ……………………………............. 33
2.10嗜中性球細胞移動分析(migration assay) …………………….. 34
2.10.1分離人類嗜中性球 …………………………………………………. 34
2.10.2細胞移動分析(migration assay) …………………………………….. 35
2.11 RT-PCR分析cytokine 的表現 ……………………………….. 37
2.11.1 RNA萃取 ……………………………………………………............ 37
2.11.2 cDNA合成:Reverse transcription …………………………………. 38
2.11.3 聚合酶連鎖反應(PCR) ……………………………………………… 39
2.11.4結果觀察 …………………………………………………………….. 41
2.12 Inhibitor assay ………………………………………………….....41
2.13西方墨點法 ………………………………………………………..42
2.13.1 細胞蛋白樣本收集 …………………………………………..............42
2.13.2電泳分析及西方墨點法 ……………………………………………... 43
2.13.3 抗體重新標定 ………………………………………………………. 44
2.14 Protease-Activated Receptor活化 ………………………………45

Ⅲ結果 ..............................................46
3.1 SPE B引發A549細胞cytokine分泌及其機制的探討 ......46
3.1.1純化SPE B 重組蛋白
3.1.2 SPE B引發人類呼吸道上皮細胞(A549)細胞分泌IL-8及IL-6
3.1.3 SPE B處理的conditioned medium可引發較多嗜中性球移動
3.1.4 SPE B引發A549細胞IL-8 mRNA表現
3.1.5 MAPK pathway 參與SPE B所引發A549細胞IL-8的分泌
3.1.6轉錄因子NF-κB參與SPE B所引發A549細胞IL-8的分泌
3.2 SPE B可能的接受器(receptor)探討 ………………………51
3.2.1純化SPE B突變株之重組蛋白
3.2.2 SPE B及其突變株蛋白分子量大小及蛋白脢活性分析
3.2.3 SPE B蛋白脢活性對A549細胞IL-8分泌之影響
3.2.4 SPE B的RGD motif對A549細胞IL-8分泌之影響
3.2.5 Protease-Activated Receptors是否為SPE B的接受器?
3.2.6 Fas是否為SPE B的接受器?
3.2.7 TLR是否為SPE B的接受器?

Ⅳ討論 …………………………………………………………56

Ⅴ參考文獻 ……………………………………………………62

圖 ………………………………………………………………70
附錄 ……………………………………………………………88
縮寫檢索表
自傳 ……………………………………………………………90
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