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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 吳世雄(Shih-Hsiung Wu) | |
| dc.contributor.author | Jui-Wen Sue | en |
| dc.contributor.author | 蘇瑞雯 | zh_TW |
| dc.date.accessioned | 2021-06-15T01:34:54Z | - |
| dc.date.available | 2009-07-23 | |
| dc.date.copyright | 2009-07-23 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-07-17 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43058 | - |
| dc.description.abstract | Klebsiella pneumonia （K. pneumoniae）克雷伯氏肺炎菌，為一常見的腸內菌科，屬於革蘭式陰性菌，經常引起醫院內群聚感染。近幾十年來，K. pneumoniae在臺灣地區造成肝膿瘍（PLA）病例增加，甚至細菌轉移至腦產生腦膜炎或眼炎。K. pneumoniae最大之特徵莫過於細菌細胞壁外層的莢膜多醣，這一層多醣構造使得細菌具有高度黏性，也是重要之毒性因子。若K. pneumoniae一旦為高黏性之菌株，就極有可能具有抵抗血清中免疫細胞之攻擊，對於宿主就會產生嚴重之影響。
而K. pneumoniae造成肝膿瘍之機制又是如何，是本篇論文所之目標。本實驗使用從肝膿瘍患者上分離到之菌株K. pneumonia NTUH-K2044，及一肝癌細胞株HepG2進行研究。研究發現，HepG2一旦被K. pneumonia NTUH-K2044感染，細胞內酪氨酸（tyrosine）磷酸化蛋白質表現量有增加的情形，而若是由pneumonia NTUH-K2044之莢膜多醣感染HepG2細胞，細胞內酪氨酸磷酸化蛋白質表現量則在2至2.5小時左右達到最高峰。而HepG2經K. pneumonia NTUH-K2044感染6小時會發現細胞數量逐漸減少、死亡，藉由流式細胞儀觀察細胞DNA，不論感染時間多寡，均維持完整之狀態，因此在此判斷細胞可能活化另外之蛋白質路徑而使細胞邁向死亡，而不是使DNA斷裂。 細胞可藉由蛋白質的磷酸化與去磷酸化轉換蛋白質的功能或活性，以達成調控細胞內訊息傳遞及生理活動，肝膿瘍的產生是否與這些路徑有關，是可以探討的方向。本研究最後希望藉由磷酸化蛋白質體之方式研究HepG2細胞在有無K. pneumonia NTUH-K2044 CPS刺激下觀察蛋白質磷酸化是否有差異，及找出相關之訊息傳遞路徑。實驗結果顯示膠內挖取得到之蛋白質並非西方墨點法所壓到之蛋白質，而以serine及threonine磷酸化之蛋白質居多，而無發現符合之酪氨酸磷酸化蛋白質。 | zh_TW |
| dc.description.abstract | Klebsiella pneumoniae, an enterobacterium, is a gram-negative strain of bacteria that belongs to enterobacteriae. K. pneumoniae is responsible for most cases of hospital-acquired pneumonia, The bacteriae often causes severe pyogenic liver abscess （PLA） that is accompanied with metastatic meningitis or endophthalmitis recently. However, it remains obscure how K. pneumoniae may become virulent in human host and result in liver abscess. It is tentative to suggest that its toxicity rises from its remarkable mucoviscosity, which is modulated by the level of external capsule polysaccharides (CPS). It is possible that once K. pneumoniae attains hypermuscosity, it could not be targeted by immune cells, and thereby leads to severe consequences.
In our study, we intend to find the missing link between the CPS of K. pneumonia and PLA pathogenesis by investigating the level and extent of protein phosphorylation in the infected HepG2 cells. We use a virulent strain K. pneumonia NTUH-K2044 which was isolated from a PLA patient in Taiwan, and a hepatoma cell line HepG2 as experiment model. When HepG2 was infected by K. pneumonia NTUH-K2044, the expression of phosphotyrosine protein was immediately upregulated. While HepG2 was infected by CPS extracted from K. pneumonia NTUH-K2044, phosphotyrosine proteins increased to optimum in 150 min. After K. pneumonia NTUH-K2044 infection for 6 hours, HepG2 died. We used a flow cytometry to identify whether cells died of apoptosis. The result showed that the DNA of cells was intact, we speculated, therefore, that cells apoptosis were activated via other pathways. Phosphorylation of proteins plays an important role in cellular process by modulating protein function and transmitting signals within cellular pathways and networks. To identify the phosphoproteins involve in the CPS infection, we use MS/MS to study the phosphoproteome of infected HepG2. Because the signal intensity phosphoserine and phosphothreonine protein was too high and put a cover over phosphotyrosin in MS/MS analysis, there was no remarkable proteins found in in-gel digestion. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T01:34:54Z (GMT). No. of bitstreams: 1 ntu-98-R96b46030-1.pdf: 2832365 bytes, checksum: ceed2b55ec183f772b0d7c67521abe25 (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | 第一章 前言
第一節 克雷伯氏肺炎菌（Klebsiella pneumonia） 與肝膿瘍（liverabscess）..........01 第二節 K. pneumonia NTUH-K2044 莢膜多醣 （CPS）............................05 第三節 真核細胞之磷酸化與磷酸化蛋白質體學08 第四節 研究動機與目的....................12 第二章 材料與方法 第一節 實驗材料..........................15 第二節 實驗方法..........................18 第三章 結果 第一節 決定Klbsiella pneumonia NTUH-K2044 之感染條件........................35 第二節 HW-65F管柱層與親和性管柱層析......37 第三節 HepG2受到K. pneumonia NTUH-K2044 及其CPS感染後酪氨酸蛋白質磷酸化 之變化............................43 第四節 經K.pneumoniae NTUH-K2044感染之HepG2 細胞凋亡觀察......................49 第五節 酪氨酸蛋白質體之研究..............53 第四章 討論 第一節 HepG2經過K. pneumonia NTUH-K2044 及其CPS 刺激後 Phosphotyrosine 蛋白質變化之探討..55 第二節 經K. pneumonia NTUH-K2044感染之HepG2 如何步向死亡......................56 第三節 K. pneumonia NTUH-K2044感染之HepG2細胞中 磷酸化蛋白質體的變化..............57 參考文獻......................59 | |
| dc.language.iso | zh-TW | |
| dc.subject | 克雷伯氏肺炎菌 | zh_TW |
| dc.subject | 莢膜多醣 | zh_TW |
| dc.subject | 肝膿瘍 | zh_TW |
| dc.subject | 磷酸化蛋白質體學 | zh_TW |
| dc.subject | capsule polysaccharide | en |
| dc.subject | liver abscess | en |
| dc.subject | phosphoproteome | en |
| dc.subject | Klebsiella pneumoniae | en |
| dc.title | 克雷伯氏肺炎菌NTUH-K2044及其莢膜多醣感染之HepG2細胞磷酸化蛋白質體之研究 | zh_TW |
| dc.title | Characterization of phosphoproteome of HepG2 cells infected by Klebsiella pneumonia NTUH-K2044 and its capsular polysaccharide. | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 邱繼輝(Kay-Hooi Khoo),花國鋒(Kuo-Feng Hua) | |
| dc.subject.keyword | 克雷伯氏肺炎菌,莢膜多醣,肝膿瘍,磷酸化蛋白質體學, | zh_TW |
| dc.subject.keyword | Klebsiella pneumoniae,capsule polysaccharide,liver abscess,phosphoproteome, | en |
| dc.relation.page | 65 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2009-07-17 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-98-1.pdf 未授權公開取用 | 2.77 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。