臺灣博碩士論文加值系統

腸球菌會引起與醫療相關的感染 (HAI)，其中包括手術傷口感染、心內膜炎、菌血症、尿路感染和腦膜炎。近年來，醫療相關的感染(HAI)的主要致病菌之一為萬古黴素抗藥性腸球菌 (VRE)，尤其是萬古黴素抗藥性腸球菌 (VRE)中，又以屎腸球菌比例為最高。本研究的目的是想要了解屎腸球菌(Enterococcus faecium)對於萬古黴素感受性的比較蛋白質體學。一、我們使用LIVE/DEAD雙螢光染色檢測萬古黴素感受性屎腸球菌(VSE)和萬古黴素抗藥性腸球菌 (VRE)受萬古黴素調控後的變化，結果顯示在1小時可以區分VRE和VSE。二、我們使用SDS-PAGE偵測屎腸球菌受萬古黴素調控一小時後的蛋白質改變，結果顯示無法清楚看到蛋白質變化。三、我們使用同位素標記相對和絕對定量方法(iTRAQ)來鑑定改變蛋白質，結果顯示出VSE有303種顯著性蛋白(向上調節蛋白質為164種、向下調節蛋白質有139種)，VRE有269 種顯著性蛋白(向上調節蛋白質為114種、向下調節蛋白質有155種)。四、VSE向上調解的132種特有蛋白質、VRE向上調解的82種特有蛋白質。透過蛋白質分析得到的結果，有助於我們瞭解萬古黴素抗藥性屎腸球菌 (VRE) 與萬古黴素感受性的屎腸球菌(VSE)在加入萬古黴素作用之後的分子機制和細胞反應。

The enterococci cause the healthcare-associated infection (HAI) including surgical wound infection, endocarditis, bacteremia, urinary tract infections, and meningitis. Recently, the vancomycin-resistant enterococci (VRE) have emerged as a major cause of HAI. In particular, the vancomycin-resistance in Enterococcus faecium is highest among all species of VRE. This study comparative proteomic on the susceptibility of Enterococcus faecium to vancomycin. First, we used LIVE/DEAD staining detecting vancomycin-susceptibility Enterococcus faecium (VSE) and vancomycin-resistant Enterococcus faecium (VRE) of vancomycin treatment. The results showed that in 1 hours can distinguish between VRE and VSE. Second, we used SDS-PASE detection Enterococcus faecium of vancomycin treatment for 1 hours. The results showed that cannot distinguish the change of protein. Third, we using isobaric tags for relative and absolute quantitation (iTRAQ) method identification change protein. The results show that the VSE has 303 significant protein (164 up regulation protein, 139 down regulated protein )
VRE has 269 significantly protein (114 up regulation proteins and 155 down regulated proteins). At last, VSE has 117 specific proteins and VRE has 82 specific proteins were found. The protein analysis results can help us understand the molecular mechanism and cellular reaction of Vancomycin-resistant Enterococcus faecium (VRE) to Vancomycin-sensitive Enterococcus faecium (VSE) by adding vancomycin.

誌謝-i
中文摘要 -iii
英文摘要 -iv
目錄 -v
表目錄 -vii
圖目錄 -viii
名詞簡寫說明-ix
第一章 緒論-1
第二章 文獻探討-2
第一節 屎腸球菌-2
一、 屎腸球菌的生長特性及環境-2
二、 屎腸球菌的基因型-3
三、 屎腸球菌感染所造成的疾病- 4
四、 腸球菌感染的流行病學-5
第二節 屎腸球菌感染治療-6
一、 屎腸球菌治療概述-6
二、 治療藥物-7
三、 萬古黴素的重要性- 7
四、 屎腸球菌藥敏試驗- 8
第三節 萬古黴素抗藥性屎腸球菌-9
一、 萬古黴素抗藥性屎腸球菌的重要性-9
二、 萬古黴素抗藥性屎腸球菌治療-9
三、 萬古黴素可變性的屎腸球菌-10
第四節 蛋白質體學的測定方法-11
一、 二維電泳法-11
二、 差異性二維電泳法-12
三、 質譜儀-13
四、 相對和絕對同位素標記分析法-14
第五節 研究動機-15
第六節 研究架構-16
第三章 研究方法-17
第一節 實驗材料與設備-17
一、 菌株-17
二、 培養基-17
三、 實驗藥品及試劑-18
四、 實驗儀器-21
第二節 實驗方法-23
一、 屎腸球菌培養-23
二、 雙螢光染色-23
三、 螢光儀定量-24
四、 蛋白質萃取-25
五、 蛋白質電泳分析-26
六、 相對和絕對同位素標記分析-28
七、 蛋白質體比對-35
第四章 研究結果-36
第一節 使用雙螢光染色法偵測屎腸球菌的死亡-36
第二節 屎腸球菌受萬古黴素調控的顯著改變蛋白質-38
第三節 萬古黴素感受性屎腸球菌受萬古黴素調控的蛋白質-40
第四節 萬古黴素抗藥性屎腸球菌受萬古黴素調控的蛋白質-42
第五節 比較萬古黴素感受性以及萬古黴素抗藥性屎腸球菌受萬古黴素調控的蛋白質差異-44
第六節 屎腸球菌受萬古黴素調控後的顯著改變蛋白質路徑-46
第五章 討論與結論-48
第一節 使用雙螢光染色法偵測屎腸球菌的死亡-49
第二節 屎腸球菌受萬古黴素調控的顯著改變蛋白質-50
第三節 比較萬古黴素感受性以及萬古黴素抗藥性屎腸球菌受萬古黴素調控的蛋白質差異-52
第四節 屎腸球菌受萬古黴素調控的改變蛋白質路徑-54
第五節 結論-55
參考文獻 -56
附錄一 蛋白質數目總整理- 127
附錄二 2018年海峽兩岸檢驗醫學論壇暨第18屆海峽兩岸檢驗醫學學術交流會議-128





表 目 錄

表一、 以iTRAQ分析經萬古黴素處理1小時的VSE和VRE概述-66
表二、 使用iTRAQ 分析VSE受萬古黴素調控所產生的164種向上調節蛋白質功能分類-67
表三、 使用iTRAQ 分析 VSE受萬古黴素調控所產生的164種向上調節蛋白質及改變倍數-68
表四、 使用iTRAQ 分析VSE受萬古黴素調控所產生的139種向下調節蛋白質功能分類-75
表五、 使用iTRAQ 分析 VSE受萬古黴素調控所產生的139種向下調節蛋白質及改變倍數76
表六、 使用iTRAQ 分析VRE受萬古黴素調控所產生的114種向上調節蛋白質功能分類-82
表七、 使用iTRAQ 分析VRE受萬古黴素調控所產生的114種向上調節蛋白質及改變倍數-83
表八、 使用iTRAQ 分析VRE受萬古黴素調控所產生的155種向下調節蛋白質功能分類-88
表九、 使用iTRAQ 分析VRE受萬古黴素調控所產生的155種向下調節蛋白質及改變倍數-89
表十、 受萬古黴素調控而向上調節的VSE蛋白質包括VSE所特有蛋白質與VRE共同的蛋白質-96
表十一、 受萬古黴素調控而向上調節的132種VSE特有蛋白質-97
表十二、 受萬古黴素調控而向上調節的VRE蛋白質包括VRE所特有蛋白質及VRE與VSE共同的蛋白質-103
表十三、 受萬古黴素調控而向上調節的82種VRE特有蛋白質-104
表十四、 VSE的303種顯著改變蛋白質功能路徑分析-108
表十五、 VRE的269種顯著改變蛋白質功能路徑分析-109

圖 目 錄

圖一、 以螢光顯微鏡觀察經萬古黴素處理VSE在0、1、2小時的變化-110
圖二、 以螢光顯微鏡觀察經萬古黴素處理VRE在0、1、2小時的變化-111
圖三、 以螢光儀偵測經萬古黴素處理VSE造成細菌死亡-112
圖四、 經萬古黴素處理VSE兩小時內的存活率-113
圖五、 經萬古黴素處理1小時的VSE和VRE蛋白質表現-114
圖六、 經萬古黴素處理1小時VSE的顯著差異蛋白質數目-115
圖七、 經萬古黴素處理1小時VSE的顯著差異蛋白質功能分析-116
圖八、 VSE受萬古黴素調控所產生的164種向上調節蛋白質功能分類-117
圖九、 VSE受萬古黴素調控所產生的139種向下調節蛋白質功能分類-118
圖十、 經萬古黴素處理1小時VRE的顯著差異蛋白質數目-119
圖十一、 經萬古黴素處理1小時VRE的顯著差異蛋白質功能分析-120
圖十二、 VRE受萬古黴素調控所產生的114種向上調節蛋白質功能分類-121
圖十三、 VRE受萬古黴素調控所產生的155種向下調節蛋白質功能分類-122
圖十四、 受萬古黴素調控而向上調節的132種VSE特有蛋白質-123
圖十五、 受萬古黴素調控而向上調節的82種VRE特有蛋白質-124
圖十六、 利用KEGG軟體分析VSE的蛋白質功能路徑-125
圖十七、 利用KEGG軟體分析VRE的蛋白質功能路徑-126

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