臺灣博碩士論文加值系統

創傷弧菌是一種嗜鹽性革蘭氏陰性桿菌，在自然環境中創傷弧菌存在於海水裏。創傷弧菌會造成人類的伺機性感染，感染包括敗血症與傷口感染。由於莢膜多醣體為潛在的毒力因子之一，與生物膜的形成有關。為了要探討莢膜多醣體對於創傷弧菌生物膜形成的影響，因此本研究採用缺乏莢膜多醣體表面因子的突變株JF046 (ΔORFVV0364) 與野生株YJ016，分析其細菌生長特性與生物膜形成量；並且以qRT-PCR測量群體感應調控子LuxR與SmcR基因的mRNA表現量，進行分析比較。結果顯示：(一)、在菌落型態上，YJ016為不透明菌落，而JF046為半透明菌落。(二)、細菌生長曲線的結果顯示，(A600) 無論是在海水環境 (經24小時培養，YJ016為1.795，JF046為1.486；p=0.006)，或淡水環境 (經24小時培養，YJ016為1.519，JF046為1.141；p=0.005)，莢膜多醣體突變株JF046的生長密度都比野生株YJ016低。(三)、在72小時培養後，莢膜多醣體的突變株JF046的生長會產生細菌聚集體，而野生株YJ016的生長則無。(四)、在生物膜形成的分析試驗中，發現生物膜形成能力（A492/A620），無論是在海水環境 (YJ016為0.91±0.15，JF046為1.50±0.23；p=0.047)，或淡水環境 (YJ016為0.50±0.05，JF046為1.26±0.20；p=0.013) ，莢膜多醣體突變株JF046的生物膜形成能力都比野生株YJ016高。(五)、在群體數量感應基因LuxR/SmcR的mRNA表現方面，分析群體數量感應調控子LuxR 與SmcR基因的表現相關性，以ANOVA回歸分析的結果顯示：在野生株YJ016 的r2 =0.885 (y=1.045x+7.798)，在突變株JF046的r2 = 0.798 (y=0.928x+25.835)。本研究綜合以上結果證明創傷弧菌莢膜多醣體表面因子ORFVV0364會影響生物膜形成及群體數量感應調控子LuxR/SmcR的表現。

Vibrio vulnificus is a halophilic gram-negative bacterial species. In nature, V. vulnificus is present in marine environment and causes opportunistic infections in humans including septicemia and wound infection. The capsular polysaccharide is one of potential virulence factors and associated with formation of biofilm. To understand the effect of capsular polysaccharide on the formation of biofilm, this study uses the acapsular strain JF046 (ΔORFVV0364) with a mutation in capsular polysaccharide surface factor. The characteristics of bacterial growth and formation of biofilm were analyzed between strains. Furthermore, the mRNA expression of quorum-sensing regulators including LuxR and SmcR were determined and compared. First, the colony of YJ016 appeared to be opaque and JF046 was translucent. Second, the results of growth curve indicated that the bacterial density for growth (A600) were lower in JF046 than those in YJ016 both in seawater environment (cultured for 24 hours, 1.795 for YJ016, 1.486 for JF046; p=0.006) and fresh water environment (cultured for 24 hours, 1.519 for YJ016, 1.141 for JF046; p=0.005), respectively. Third, the bacterial aggregates were existed in JF046 and absent in YJ016. Fourth, the ability for formations of biofilm (A492/A620) in acapsular mutant JF046 were higher than those in wild type YJ016 both in seawater environment (0.91±0.15 for YJ016, 1.50±0.23 for JF046; p=0.047) and fresh water (0.50±0.05 for YJ016, 1.26±0.20 for JF046; p=0.013). Fifth, the expressions of quorum-sensing regulators, LuxR and SmcR, were compared between JF046 and YJ016. The results form regression analysis revealed that the correlation between mRNA expressions of LuxR and SmcR were r2=0.885 (y=1.045x+7.798) in YJ016 and r2=0.798 (y=0.928x+25.835) in JF046, respectively. In conclusion, the results demonstrated that the ORFVV0364 for capsular polysaccharide surface factor of Vibrio vulnificus is associated with formation of biofilm and quorum-sensing regulators expression.

目錄
致謝 i
摘要 iii
Abstract v
目錄 vii
表目錄 xi
圖目錄 xii
名詞縮寫說明 xiv
第一章 ?論 1
第一節 創傷弧菌特性 1
一、創傷弧菌的生長背景與感染 1
二、創傷弧菌傳播途徑 2
三、創傷弧菌易感宿主 2
四、創傷弧菌感染症狀 3
五、創傷弧菌的毒力因子 4
第二節生物膜 6
一、生物膜的形成 6
二、生物膜的致病機制 7
第三節 創傷弧菌與群體數量感應調控子 8
一、調控子LuxR 8
二、調控子SmcR 9
三、群體數量感應系統 (Quorum-sensing system) 10
第四節 莢膜多醣體對於創傷弧菌形成生物膜的影響 12
一、創傷弧菌莢膜多醣體的角色 12
二、創傷弧菌莢膜多醣體與生物膜形成的關係 12
第五節 研究動機與目的 14
第六節 研究架構圖 15
第二章 材料與方法 16
第一節 實驗材料與設備 16
一、菌株 16
二、培養基與藥品試劑 17
(一)、培養基成分 17
1. LB (Luria-Bertani broth) 17
2. LB agar (Luria-Bertani agar) 17
3.ASWLB (Artificial Seawater Luria-Bertani) 17
(二)、實驗藥品及試劑 18
1.配製人工海水之藥品 18
2.RNA萃取藥品 18
3.反轉錄聚合?○s鎖反應試劑 19
4.即時定量聚合?○s鎖反應試劑 19
5.Primer序列 (資料來源NCBI) 20
三、實驗儀器 27
(一)、反轉錄聚合?○s鎖反應儀器 27
(二)、即時定量聚合?○s鎖反應儀器 (Fast real-time PCR) 27
(三)、生物膜形成的分析儀器（Biofilm assay) 27
第二節 實驗方法 28
一、細菌培養與保存 28
二、生物膜形成分析 (Biofilm assay) 28
三、生長曲線分析 29
四、RNA萃取與反轉錄作用 30
(一)、RNA萃取 30
(二)、RNA反轉錄作用 31
五、引子設計與合成 32
六、即時定量反轉錄聚合?○s鎖反應分析 33
七、統計 34
第三章 研究結果 35
第一節分析創傷弧菌野生株YJ016與突變株JF046的生長情形 35
一、菌落型態及LB的生長情形 35
二、細菌的生長曲線 35
第二節分析創傷弧菌野生株YJ016與突變株JF046生物膜形成 38
第三節分析創傷弧菌野生株YJ016與突變株JF046群體數量感應調控子基因mRNA的表現量 39
一、群體數量感應調控子LuxR 39
二、群體數量感應調控子SmcR 39
三、群體數量感應調控子的表現相關性 40
第四章 討論 41
第一節分析創傷弧菌野生株YJ016與突變株JF046的生長 42
第二節分析創傷弧菌野生株YJ016與突變株JF046生物膜形成 44
第三節分析創傷弧菌野生株YJ016與突變株JF046群體數量感應調控子基因mRNA的表現量 45
參考文獻 47
附錄 76
附錄一、創傷弧菌野生株YJ016的基因體 76
附錄二、創傷弧菌淺在的毒力因子 77
附錄三、創傷弧菌突變株 JF046 (ΔORFVV0364) 的來源 78
附錄四、VV0364的相關資料 (NCBI) 79
附錄五、Vibrio harveyi 群體數量感應調控系統 81
附錄六、Vibrio vulnificus 群體數量感應調控系統 82
附錄七、Lux family 83
附錄八、群體數量感應調控子mRNA表現 84
附錄九、群體數量感應調控子LuxR與SmcR基因的ANOVA回歸分析 85
附錄十、海水組成分佈圖 87
附錄十一、人工海水配方表 88
附錄十二、研討會壁報發表 89
附錄十三、其他研討會壁報發表 90




































表目錄

表一、實驗當中所使用的引子 56



圖目錄

圖一、創傷弧菌JF046及YJ016菌落型態 57
圖二、創傷弧菌細菌聚集體的形成 58
圖三、創傷弧菌YJ016的生長曲線 59
圖四、創傷弧菌JF046的生長曲線 60
圖五、創傷弧菌YJ016與JF046在LB培養基的生長情形 61
圖六、創傷弧菌YJ016與JF046在ASWLB培養基的生長情形 62
圖七、創傷弧菌YJ016與JF046在LB或ASWLB培養基的生長情形 63
圖八、創傷弧菌YJ016培養於不同環境下的對數期 64
圖九、創傷弧菌JF046培養於不同環境下的對數期 65
圖十、創傷弧菌YJ016與JF046培養於LB培養基的對數期 66
圖十一、創傷弧菌YJ016與JF046培養於ASWLB培養基的對數期 67
圖十二、創傷弧菌YJ016與JF046培養於不同環境下的對數期 68
圖十三、創傷弧菌生物膜的形成 69
圖十四、創傷弧菌生物膜形成的比較 70
圖十五、創傷弧菌YJ016及JF046在群體數量感應調控子LuxR基因之
mRNA之相對表現(％) 71
圖十六、創傷弧菌YJ016及JF046在群體數量感應調控子SmcR基因之
mRNA之相對表現(％) 72
圖十七、 創傷弧菌YJ016在群體數量感應調控子LuxR/SmcR基因之
mRNA之相對表現(％)...................................................................73
圖十八、 創傷弧菌JF046在群體數量感應調控子LuxR/SmcR基因之
mRNA之相對表現(％)...................................................................74
圖十九、 創傷弧菌野生株YJ016與突變株JF046在群體數量感應調控子
LuxR與SmcR基因經 ANOVA迴析…........................................75

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