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研究生:羅世傑
研究生(外文):LO,SHIH-CHIEH
論文名稱:探討吞噬細胞對創傷弧菌胞外多醣體產生的免疫反應
論文名稱(外文):Studies on immune responses of macrophage to the extracellular polysaccharide of Vibrio vulnificus
指導教授:黃小萍黃小萍引用關係
指導教授(外文):HUANG,SHIAO-PING
口試委員:賴志河曹德安
口試委員(外文):LAI,CHIH-HOTSAO,DER-AN
口試日期:2016-06-15
學位類別:碩士
校院名稱:輔英科技大學
系所名稱:醫學檢驗生物技術系碩士班
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:76
中文關鍵詞:發炎反應M1-M2 巨噬細胞細胞激素胞外多醣體創傷弧菌
外文關鍵詞:InflammationM1-M2 MacrophagesCytokinesExtracellular PolysaccharideVibrio vulnificus
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在先天性免疫反應中,發炎的重要性在於它是面臨感染或刺激時的第一個反應。在此,巨噬細胞扮演著重要的角色。透過不同訊號路徑的活化作用後,巨噬細胞能發展出功能迥異的兩個表現型,包括經典型巨噬細胞(M1)和替代型巨噬細胞(M2)。細菌的多醣體,目前已被推定具有刺激巨噬細胞參與發炎反應的能力。本研究比較了三個創傷弧菌菌株,包括野生型菌株(YJ016)、突變型菌株(JF045)和互補型菌株(JF049)所誘導的發炎相關細胞激素基因表現,以了解這些抗原是否與發炎相關細胞激素的誘導有關。本研究使用創傷弧菌菌株,包括野生型菌株(YJ016)、突變型菌株(JF045)和互補型菌株(JF049)感染小鼠巨噬細胞RAW264.7細胞。首先測定每個菌株的生長曲線,其結果應用於計算感染到小鼠RAW264.7細胞的感染劑量。接著在感染0.5、1、1.5和2小時的時間萃取細胞RNA,並經反轉錄和即時PCR,測量以下訊號,包括IL-1β、IL-6、TRAF6、TLR-2、TLR-4、Arg-1、TNF-α、IL-4、IFN-γ和IL-10的基因表現量。結果顯示創傷弧菌的野生型菌株(YJ016)誘導RAW264.7細胞的TLR-2、TLR-4、TNF-α、IL-1β、IL-4、IL-10和Arg-1表現性上升,並有一定的時間與次序。在突變型菌株(JF045)我們觀察到在感染早期有發炎反應,並與野生型菌株(YJ016)類似,具有訊號表現上升的現象。在互補型菌株(JF049)觀察到誘發RAW264.7細胞表現TLR-2、TLR-4、TNF-α、IL-1β 及 IL-10上升,而IL-4 及 Arg-1表現性下降。此外,我們觀察到互補型菌株(JF049)的訊號調控較野生型菌株(YJ016)慢。最後,本研究的結果有助於我們了解EPS對M1-M2巨噬細胞的調控與其時間與次序的現象。
As an essential component of innate immunity, inflammation is one of the first responses to infection or irritation. In the responses, macrophages play a crucial role. It can develop distinct functional phenotypes via undergoing different phenotypic polarization, including classically activated macrophages (M1) and alternatively activated macrophages (M2). The bacterial polysaccharide have been postulated that with significant of stimulate macrophages, which are involved in the inflammatory response. This study compared the induction of inflammation-related cytokines among the three strains of Vibrio vulnificus including the wild-type strain (YJ016), the capsular polysaccharide (CPS) mutant strain (JF045) and the complementary strain (JF049) to understand whether these antigens contributes to the induction of inflammatory cytokines and the mechanism of pathogenesis. The bacteria strains including the wild-type strain (YJ016), the CPS mutants (JF045), and the complementary strain (JF049) were applied in the infection of Vibrio vulnificus strains to RAW264.7 cells. This study aims to measure each bacteria of growth curves, the results were subjected for counting the multiplication of infection to RAW264.7 cells. Then the extracted cellular RNA at the infection for 0.5, 1, 1.5 and 2 h were applied in the reverse transcription and real-time PCR for measuring the expression of signals including IL-1β, IL-6, TRAF6, TLR-2, TLR-4, Arg-1, TNF-α, IL-4, IFN-γ and IL-10. The results showed that the Vibrio vulnificus wild-type strain (YJ016) induced RAW264.7 cells with upregulation of TLR-2, TLR-4, TNF-α, IL-1β, IL-4, IL-10, and Arg-1 in a time dependent manner. With CPS mutants (JF045), we observed an inflammatory reaction in the early stage of infection, and with the similar upregulation as the wild-type strain. The complementary strain (JF049) induced RAW264.7 cells with upregulation of TLR-2, TLR-4, TNF-α, IL-1β and IL-10 and downregulation of IL-4 and Arg-1. Besides, we observed that the signal regulations of the complementary strain (JF049) were later than those of wild-type strain (YJ016). Eventually, the results help us to understand the effect of EPS on the M1-M2 polarization of macrophages and the time dependent manner.
誌謝 i
中文摘要 iii
英文摘要 v
目錄 vii
表目錄 ix
圖目錄 x
符號說明 xi
第一章 緒論 1
第二章 文獻探討 2
第一節 炎症(Inflammation) 2
第二節 巨噬細胞 3
第三節 細胞激素(Cytokines) 5
第四節 介白質-1 (IL-1) 6
第五節 腫瘤壞死因子-阿爾法(TNF-α) 7
第六節 介白質-6 (IL-6) 8
第七節 腫瘤壞死因子受器相關因子蛋白第六型(TRAF6) 9
第八節 精胺酸酶1(Arg-1) 10
第九節 介白質-4 (IL-4) 11
第十節 介白質-10(IL-10) 12
第十一節 干擾素-伽馬(IFN-γ) 13
第十二節 類鐸受器(TLR) 14
第十三節 創傷弧菌 15
第十四節 胞外多醣體 16
第三章 研究材料與方法 17
第一節 實驗材料與設備 17
一、菌株 17
二、細胞 19
三、培養基 19
第二節 研究方法 21
一、細胞培養與保存 21
二、菌種培養與保存 22
三、感染後的細胞RNA之萃取 25
四、cDNA之合成 27
五、即時反轉錄聚合酶連鎖反應 (RT-qPCR) 28
第四章 研究結果 29
第一節 創傷弧菌在剛果紅培養基的菌落 29
第二節 YJ016誘發細胞TLR2、TLR4及TRAF6基因表現 30
第三節 YJ016誘發細胞之IL-4及IL-10基因表現 31
第四節 YJ016誘發細胞之IFN-γ基因表現 32
第五節 YJ016誘發細胞之Arg-1基因表現 33
第六節 YJ016誘發細胞之TNF-α、IL-1β及IL-6基因表現 34
第七節 JF045誘發細胞之TLR2、TLR4及TRAF6基因表現 35
第八節 JF045誘發細胞之IL-4及IL-10基因表現 36
第九節 JF045誘發細胞之IFN-γ基因表現 37
第十節 JF045誘發細胞之Arg-1基因表現 38
第十一節 JF045誘發細胞之TNF-α、IL-1β及IL-6基因表現 39
第十二節 JF049誘發細胞之TLR2、TLR4及TRAF6基因表現 40
第十三節 JF049誘發細胞之IL-4及IL-10基因表現 41
第十四節 JF049誘發細胞之IFN-γ基因表現 42
第十五節 JF049誘發細胞之Arg-1基因表現 43
第十六節 JF049誘發細胞之TNF-α、IL-1β及IL-6基因表現 44
第五章 討論與結論 45
參考文獻 48

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