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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王錦堂(Jin-Town Wang) | |
| dc.contributor.author | Hung-Chuan Chiu | en |
| dc.contributor.author | 邱泓銓 | zh_TW |
| dc.date.accessioned | 2021-06-15T02:48:37Z | - |
| dc.date.available | 2012-09-15 | |
| dc.date.copyright | 2009-09-15 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-08-06 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44278 | - |
| dc.description.abstract | 在醫院中，細菌藉由消毒不完全的內視鏡而傳播到下一位使用者的是常見的情況。一般而言，內視鏡的使用後的處理包括事先的清潔 (manual pre-cleaning) 與利用戊二醛 (glutaraldehyde) 作浸泡消毒，但是這個過程仍舊無法將細菌完全的殺死，有些對於清潔劑或是有機溶劑耐受性的細菌會經由未知的機制在此過程中存活。有鑑於此，我們的利用0.1 %戊二醛的培養基去篩選結果包含有幽門螺旋桿菌NTUH-C1 DNA片段的表現基因庫的結果，我們總共找到七個菌落，經由序列比對的結果顯示，其中五顆菌落的基因比對為涵蓋HP0929-HP0930的區域，另外兩顆菌落的基因序列比對為imp/ostA這個基因，此基因片段經表現蛋白並製備多株抗體，使用OstA多株抗體去偵測正常株以及突變株的細胞萃取物可以發現imp/ostA在幽門螺旋桿菌中不為一個必要的基因，有機溶劑測試的結果顯示imp/ostA在有機溶劑抵抗性上所扮演的角色，抗生素測試的結果顯示imp/ostA的突變株對於β-lactam 以及疏水性的抗生素具有敏感性，N-phenylnapthylamine (NPN) assay的結果顯示，imp/ostA突變株的外膜通透能力與正常株比較起來約增加了50% (P< 0.001) 。
我們想更近一步了解戊二醛耐受性的作用機制，從1991-2000年台大醫院內視鏡鏡檢所分離出來的49株臨床菌株中，我們測試其對於戊二醛的最小抑菌濃度以及與imp/ostA基因表現的相互關係。在RNA層次上，imp/ostA的基因表現量，在戊二醛最小抑菌濃度4~10μg/ml的菌株明顯高於戊二醛最小中抑菌濃度1~3 μg/ml的菌株，此外，我們挑選了一株戊二醛耐受性較高的菌株NTUH-S1，並且以戊二醛誘導其基因表現，經由基因微陣列的結果，我們發現有40個基因表現量上升，而有31個基因表現量下降，在表現量上升的基因之中，除了之前所鑑定的imp/ostA這個基因外，我們還發現在在功能上與imp/ostA同為假設性脂多醣生合成有關的基因的msbA進行研究，研究結果顯示，imp/ostA以及msbA的突變株，對於戊二醛或是疏水性抗生素的敏感性與正常株比較起來，都有增加的趨勢，而imp/ostA與msbA雙重突變株對於戊二醛以及疏水性抗生素產生高度敏感性。銀染的結果則顯示了，脂多醣的含量在imp/ostA以及msbA突變株中都有減少的情形，而在imp/ostA與msbA雙重突變株中則大為減少。外膜通透性的實驗說明了，imp/ostA與msbA兩個基因的雙重突變會造成外膜對於毒物通透性大為增加。Ethidium bromide累積測試的實驗顯示了，MsbA在功能上面，牽涉到將疏水性藥物從菌體內排出到菌體外的功能。 總而言之，在這些臨床菌株中，經由戊二醛的刺激，imp/ostA與msbA基因的表現與戊二醛的耐受性之間是有關聯性的，Imp/OstA與MsbA在疏水性藥物的耐受性與脂多醣的生合成上具有協同作用並且扮演重要的角色。 | zh_TW |
| dc.description.abstract | Pre-cleaning and soaking in glutaraldehyde is the necessary procedure to disinfect endoscopes. However, some chemical solvent tolerance bacteria may survive after incomplete endoscopic disinfection. Basing on this, we selected the expression library of H. pylori strain NTUH-C1 with 0.1 % glutaraldehyde obtained seven colonies. After DNA sequencing, DNA fragments of five colonies were identical (homologous to 2.2 kb DNA fragment containing HP0929 and HP0930 of H. pylori strain 26695) and DNA fragments of the other two of the seven colonies were identical (homologous to HP1216 of H. pylori strain 26695). OstA recombinant protein was expressed and used to generate anti-OstA polyclonal antibody. Using OstA polyclonal antibody against cell lysate of wild-type and imp/ostA mutant showed that it is not essential in H. pylori. Organic solvent tolerance assay demonstrated the role of imp/ostA in organic solvent tolerance. Antibiotic susceptibility test showed that the mutation of imp/ostA was susceptible to hydrophobic and β-lactam antibiotics. NPN assay demonstrated that the level of outer membrane permeability was increased 50 % in mutant strain comparing to wild-type strain (P< 0.001).
We sought to further clarify the mechanism of glutaraldehyde resistance. The minimal inhibitory concentrations of glutaraldehyde and imp/ostA RNA expression in 49 clinical isolates from National Taiwan University Hospital were determined. After glutaraldehyde induction, RNA expression was higher for strains with minimal inhibitory concentrations of 4~10 μg/ml than for strains with minimal inhibitory concentrations of 1~3 μg/ml. When a microarray was used to study full-genome expression of a strain NTUH-S1 with minimal inhibitory concentration of 6 | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T02:48:37Z (GMT). No. of bitstreams: 1 ntu-98-D92445003-1.pdf: 1330039 bytes, checksum: 8a63291d9ac2b37393cdd4220fc7becb (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | 中文摘要………………………………………………………………………….…...1
英文摘要…………………………………………………………………………...…3 第一章 緒論……………………………………………………………………......5 第二章 材料及方法 1.細菌株 (strains) ………………………………………………………...……12 2.質體 (plasmids) ………………………………………………………...……13 3.引子 (primers) …………………………………………………………...…..14 4.培養基 (medium) ………………………………………………………...…..16 5.抗生素 (antibiotics) …………………………………………………......…..17 第三章 實驗方法 1 以戊二醛篩選含有幽門螺旋桿菌NTUH-C1基因片段的表現 基因庫…………………………………………………………………………18 2 決定對於戊二醛有抵抗性基因片段之最小抑菌濃度…………….......……18 3 建構幽門螺旋桿菌imp/ostA 基因剔除的突變株…………………...….….18 4 建構幽門螺旋桿菌imp/ostA 整段基因剔除的突變株……………...……..19 5基因補回 (complementation) imp/ostA突變株……………….……........19 6 建構幽門螺旋桿菌msbA 整段基因剔除的突變株………………………...19 7基因補回msbA突變株……………………….……………………….…...20 8 建構幽門螺旋桿菌imp及msbA 整段基因剔除的雙重突變株……...…...20 9決定幽門螺旋桿菌對於戊二醛的最小抑菌濃度…………………...…..21 10製備OstA多株抗體………………………….………………..…….............21 11有機溶劑耐受性測試…………………………………………….……...……22 12抗生素耐受性測試…………………………………………...………………23 13 N-phenylnapthylamine 測試…………………………………………….....23 14 狹縫點墨法 (slot-blot hybridization)…...…………………………….......23 15 抽取細菌的RNA並以 quantitative real-time PCR分析…………..….…24 16 西方點墨法分析細胞萃取物………………………………………….........24 17 以基因微陣列分析戊二醛耐受性菌株中被戊二醛誘導 表現的基因……………………………………………………………..........25 18 各突變株生長曲線測試………...............................................................25 19測試正常株及各突變株對於疏水性抗生素的最小抑菌濃度…….…........25 20蛋白質分解酵素 (proteinase-K) 處理幽門螺旋桿菌………………….26 21利用anti-Lewis antibody 偵測幽門螺旋桿菌的脂多醣…………...…......26 22測試正常株及各突變株對於外膜通透的情況………………………….….26 23測試正常株、msbA突變株及msbA回復株對於ethidium bromide 累積的情況.…………………………………………….…………...…..…..27 第四章 實驗結果 1以戊二醛篩選含有幽門螺旋桿菌基因片段之表現基因庫……………….…28 2 Imp/ostA在幽門螺旋桿菌對於戊二醛抵抗能力的影響…………………....29 3 Imp/ostA在幽門螺旋桿菌中不為生長必要 (non-essential) 的基因……29 4 Imp/ostA在幽門螺旋桿菌對於正己烷耐受性的影響…………………….…29 5 Imp/ostA突變使外膜通透性增加，喪失了對於抗生素耐受性………….…30 6 Imp/ostA突變株導致外膜通透性的增加…………………………..…….…..30 7 由台大醫院49株臨床菌株所造成感染者之消化道疾病 分佈與對於戊二醛之最小抑菌濃度…………………………………………31 8 經戊二醛處理後，imp/ostA基因與蛋白質表現的情況……….................31 9 基因微陣列分析戊二醛誘導前後幽門螺旋桿菌NTUH-S1 表現的基因…………………………………………………………….….…..32 10以狹縫點墨法再次確認msbA基因在戊二醛耐受菌株中的表現…….….33 11 經戊二醛處理後，在轉錄上面，imp/ostA對於msbA的影響…...….…….34 12 正常株及各突變株對於戊二醛的最小抑菌濃度…………………….….…34 13 正常株及各突變株對於疏水性抗生素的最小抑菌濃度…………….….…34 14 在正常株及各突變株中脂多醣的含量的多寡………………………..……36 15 以ethidium bromide當做螢光標的測量外膜通透性的情況……….……37 16 正常株及各突變株對於ethidium bromide累積的情況…………….……37 第五章 討論………………………………………………………………….….....39 第六章 參考文獻…………………………………………………………….….....68 第七章 附錄……………………………………………………………………….73 表目錄 表一: 戊二醛抵抗性的基因片段之最小抑菌濃度…………….……….………….44 表二: 幽門螺旋桿菌對於戊二醛之最小抑菌濃度…………………….….......45 表三: 幽門螺旋桿菌對於不同抗生素之最小抑菌濃度(μg/ml)…………….......46 表四: 從1991到2000年在台大醫院所分離出49株臨床菌株所造成 的消化道疾病與其最小抑菌濃度…………………………………............47 表五: 戊二醛處理後表現量上升之基因…………………………………………...48 表六: 戊二醛處理後表現量下降之基因…………………………………………...50 圖目錄 圖一: 膠凝電泳法與西方點墨法鑑定幽門螺旋桿菌中Imp/OstA蛋白質…..…...52 圖二: 幽門螺旋桿菌NTUH-C1在不同濃度的正己烷下的生長情況……….......53 圖三: 在含有正己烷揮發氣體下幽門螺旋桿菌NTUH-C1的生長情況…….......54 圖四: 外膜通透性測試……………………………………...…………………...….55 圖五: 由台大醫院49株臨床菌株對於戊二醛之最小抑菌濃度………....…..…...56 圖六: imp/ostA基因經戊二醛處理48小時後表現的情況………………...…......57 圖七: Imp/OstA蛋白質經戊二醛處理48小時後表現的情況…………..……......58 圖八: 以狹縫點墨法再次確認msbA基因經戊二醛處理48小時後表現的情況………………………………………………………….............59 圖九: 經戊二醛處理後，以狹縫點墨法觀察imp/ostA在轉錄上面對於msbA的影響……………………………………………………………………………..60 圖十: 各菌株於含有10 %胎牛血清的布魯塞爾培養液之生長測試……………..61 圖十一: 各菌株對於戊二醛的最小抑菌濃度………………………….…………..62 圖十二: 各菌株對於疏水性抗生素的最小抑菌濃度………………………………63 圖十三: 各菌株對於ethidium bromide的最小抑菌濃度…………………………64 圖十四:各菌株株中脂多醣的含量的多寡…………….…….…......…...65 圖十五: 以ethidium bromide觀察imp/ostA以及msbA這兩個基因對於外膜通透性的影響………………………...……………….….....…..66 圖十六: 正常株、msbA突變株及msbA基因補回株對於ethidium bromide 累積的情況…………………………..………………………….….…....…67 | |
| dc.language.iso | zh-TW | |
| dc.subject | msbA | zh_TW |
| dc.subject | 幽門螺旋桿菌 | zh_TW |
| dc.subject | 戊二醛 | zh_TW |
| dc.subject | N-phenylnapthylamine | zh_TW |
| dc.subject | imp/ostA | zh_TW |
| dc.subject | Helicobacter pylori | en |
| dc.subject | imp/ostA | en |
| dc.subject | N-phenylnapthylamine | en |
| dc.subject | gluteraldehyde | en |
| dc.subject | msbA | en |
| dc.title | 幽門螺旋桿菌imp/ostA與msbA基因功能與抵抗疏水性抗生素機制探討 | zh_TW |
| dc.title | Characterization of imp/ostA and msbA functions and the mechanism in hydrophobic drugs resistance of Helicobacter pylori | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 鄧麗珍(Lee-Jeng Teng),李財坤(Tsai-Kun Li),王雯靜(Wen-ching Wang),董馨蓮(Shin-Lian Doong) | |
| dc.subject.keyword | 幽門螺旋桿菌,戊二醛,N-phenylnapthylamine,imp/ostA,msbA, | zh_TW |
| dc.subject.keyword | Helicobacter pylori,gluteraldehyde,N-phenylnapthylamine,imp/ostA,msbA, | en |
| dc.relation.page | 76 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2009-08-06 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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