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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張震東 | |
dc.contributor.author | Yong-Tzuo Lai | en |
dc.contributor.author | 賴勇佐 | zh_TW |
dc.date.accessioned | 2021-06-16T22:59:14Z | - |
dc.date.available | 2012-08-15 | |
dc.date.copyright | 2012-08-15 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-08-08 | |
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Brain Res Mol Brain Res, 1999. 64(1): p. 24-33. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64784 | - |
dc.description.abstract | 組織缺血與再灌流(ischemia/reperfusion)是指血液供應受到阻礙與隨後的血流恢復,這種現象帶給細胞組織許多負面影響,其中粒線體失去正常功能而導致的氧化壓力將帶給細胞嚴重的傷害,在動物模式中發現外加高於生理濃度的丙酮酸可以有效的救援氧化壓力帶來的傷害。以往對於中間代謝的觀點只是提供細胞養分以及生合成的材料,然而,越來越多的證據顯示有些代謝物質可能扮演訊息傳遞的角色,這些代謝物質可能會藉著與目標蛋白的直接作用而影響其功能,為了驗證此觀點,本實驗室發展了能夠鑑定與丙酮酸直接作用的蛋白質體的方法學(AETHIC),本研究利用AETHIC與代謝體分析,探討外加的丙酮酸對於HEK細胞抗氧化能力的影響,主要針對我們鑑定到的蛋白:檸檬酸合成酶以及硫氧還蛋白還原酶(TrxR)。前者與異檸檬酸脫氫酶(NADP+-dependent isocitrate dehydrogenase, NADP+-IDH)所構成的代謝途徑可以產生細胞還原力的來源NADPH,而後者與抗氧化能力有直接的關係。
在代謝體分析中顯示丙酮酸加入HEK細胞培養液中導致檸檬酸循環流量的提高,另外NADPH/NADP+與NADH/NAD+也有明顯的增加,酵素活性分析結果顯示相同的處理下檸檬酸合成酶活性降低,所以檸檬酸合成酶活性的下降可看做對TCA cycle的調控。接著觀察檸檬酸合成酶的下游代謝:異檸檬酸脫氫酶,在in cellulo活性測試發現丙酮酸可以增加異檸檬酸脫氫酶的活性,但在in vivo實驗中,只在較丙酮酸高濃度下才觀察到,故推測細胞內有其他的機制來調控異檸檬酸脫氫酶活性。在硫氧還蛋白還原酶的研究中,觀察到氧化壓力會導致此蛋白的失活,但丙酮酸可以快速的恢復其活性至正常值,所以推測丙酮酸具有保護硫氧還蛋白還原酶的功效。由以上實驗顯示,丙酮酸對於細胞抗氧化壓力是有幫助的,可以快速增加細胞的還原能力,同時也有維持TrxR/Trx還原系統的功能。 | zh_TW |
dc.description.abstract | Ischemia/reperfusion is a pathophysiological condition of which blood flow is blocked and then restored. It will cause profound pathological effects such as oxidative stress caused by the mitochondria dysfunction. In some animal models, supplement of pyruvate effectively mitigates ischemia/reperfusion injury. There is growing evidence that some metabolites may play a new role as a signaling messenger and regulate the function of proteins in addition to their traditional roles of energy supplement and biosynthesis. To verify this, our laboratory developed a method for identification of pyruvate-interacting proteome (AETHIC). And the purpose of this study is to investigate the role of pyruvate in counteracting oxidative stress. According to AETHIC and metabolome results, we focused on citrate synthase and thioredoxin reductase (TrxR). The former enzyme and NADP+-dependent isocitrate dehydrogenase (NADP+-IDH) contribute a pathway of NADPH production, and the latter, TrxR, is directly associated with anti-oxidative function.
The flux of TCA cycle, NADPH/NADP+ and NADH/NAD+ increased after addition of pyruvate to HEK cell. Also, the activity of citrate synthase decreased under the same treatment, which indicates the regulatory role of pyruvate in TCA cycle. Next, I measured the activity of NADP+-IDH and found that pyruvate increased the activity of IDH in vitro but was less effective in cellulo. The activity of TrxR was significantly decreased under H2O2 treatment, but pyruvate completely recovered the activity to normal levels. Consequently, it is suggested that pyruvate may protect TrxR and restore its activity under oxidative stress. Above all, pyruvate rescue cells from oxidative stress by elevation of the reducing power of cells and sustaining the TrxR/Trx system. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T22:59:14Z (GMT). No. of bitstreams: 1 ntu-101-R99b46003-1.pdf: 1418432 bytes, checksum: e8dbceb105f069f3ec129870d44043a6 (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | 致謝.................................................................................................................................i
中文摘要........................................................................................................................ii 英文摘要.......................................................................................................................iii 縮寫表...........................................................................................................................iv 緒論................................................................................................................................1 第一節 組織缺血與再灌注(Ischemia/Reperfusion)造成細胞氧化壓力............1 第二節 丙酮酸在缺氧及再灌注損傷之救援效果及其參與的訊息傳遞........1 第三節 細胞調節氧化壓力與硫氧還蛋白/硫氧還蛋白還原酶系統(Thioredoxin /Thioredoxin reductase system).......................................................3 第四節 硫氧還原蛋白/硫氧還原蛋白系統對細胞存活之影響與外界的調控.4 第五節 丙酮酸在代謝功能外可能扮演的調控功能..........................................5 第二章 實驗材料與方法............................................................................................7 第一節 實驗材料..................................................................................................7 第二節 細胞培養..................................................................................................7 第三節 重組人類異檸檬酸脫氫酶 (Isocitrate Dehydrogenase, IDH)蛋白之取得............................................................................................................................7 第一項 引子設計 (Primer Designing).........................................................7 第二項 聚合酶連鎖反應 (Polymerase Chain Reaction).............................7 第三項 基因轉殖 (Cloning)........................................................................8 第四項 重組蛋白質表現與純化..................................................................8 第四節 蛋白質實驗方法......................................................................................8 第一項 丙酮酸直接作用之蛋白質體方法學..............................................8 第二項 BCA 蛋白質定量............................................................................9 第三項 SDS聚丙烯醯胺凝膠電泳 (SDS Poly-acrylamide Gel Electrophoresis, SDS-PAGE) ........................................................................9 第四項 蛋白質電泳膠片染色與銀染........................................................10 第五項 電泳轉移 (Electrophoretic Transfer)............................................10 第六項 西方轉漬法 (Western Blotting)....................................................11 第七項 試管內硫氧還蛋白還原酶活性測試............................................11 第八項 試管內檸檬酸合成酶活性測試 (in vitro citrate synthase activity assay)……………………………………………………………………….11 第九項 試管內異檸檬酸脫氫酶活性測試 (In Vitro Isocitrate Dehydrogenase Activity Assay)……………………………………..……..11 第五節 細胞學實驗方法....................................................................................12 第一項 繼代培養........................................................................................12 第二項 以H2O2處理細胞造成氧化壓力之條件.......................................12 第三項 細胞均質液製備 (Whole Cell Lysate Preparation)......................12 第四項 細胞存活率分析 (MTT Assay)....................................................12 第六節 細胞內代謝體分析…............................................................................13 第三章 實驗結果......................................................................................................14 第一節 丙酮酸處理增強細胞對抗氧化壓力....................................................14 第二節 丙酮酸處理影響HEK細胞代謝體......................................................14 第三節 與丙酮酸直接作用之蛋白質體鑑定....................................................15 第四節 丙酮酸處理對粒線體中Citrate Synthase的活性影響.........................15 第五節 丙酮酸處理對HEK細胞IDH活性的變化...........................................16 第六節 丙酮酸處理對HEK細胞TrxR活性的變化.........................................16 第七節 氧化壓力對TrxR活性造成明顯的降低..............................................17 第八節 氧化壓力下丙酮酸對於TrxR活性的恢復效果..................................17 第四章 討論................................................................................................................19 第一節 丙酮酸處理提高HEK細胞內NADPH ..............................................19 第二節 結合代謝體與酵素活性分析討論丙酮酸對於細胞的影響................20 第三節 氧化壓力對於TrxR的影響以及丙酮酸恢復TrxR活性的效果.........21 第四節 丙酮酸對HEK細胞抗氧化能力影響..................................................21 第五章 結語................................................................................................................24 第六章 實驗結果圖表................................................................................................25 圖一 丙酮酸處理可以保護氧化壓力下的HEK 293細胞................................25 圖二 (A) 丙酮酸及葡萄糖對細胞代謝之影響................................................26 (B) 丙酮酸對細胞NADH及NADPH代謝之影響...............................26 表一 丙酮酸對細胞NADH及NADPH代謝之影響......................................26 圖三 (A)(B) 丙酮酸對Citrate Synthase活性之影響.......................................28 圖四 (A)(B)(C)(D) 丙酮酸對Isocitrate Dehydrogenase活性之影響............29 圖五 (A)(B)丙酮酸對Thioredoxin Reductase活性之影響.............................32 圖六 (A) 過氧化氫處理對Thioredoxin Reductase活性之影響.....................33 圖六 (B)(C) 過氧化氫處理對Thioredoxin Reductase蛋白質量之影響…....33 圖七 (A)(B) 過氧化氫處理對Thioredoxin Reductase活性之影響................34 圖七 (C)(D) 過氧化氫處理對Thioredoxin Reductase蛋白質量之影響........34 圖八 丙酮酸解救氧化壓力下的HEK 293細胞之效果...................................36 參考文獻......................................................................................................................37 附錄..............................................................................................................................42 附圖一 AETHIC各管柱沖提結果銀染圖.........................................................42 附圖二 AETHIC蛋白鑑定結果........................................................................43 附圖三 本研究推測的NADPH產生途徑.........................................................44 | |
dc.language.iso | zh-TW | |
dc.title | 探討丙酮酸對HEK 293細胞抗氧化能力之影響 | zh_TW |
dc.title | The roles of pyruvate in anti-oxidation in HEK 293 cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 李明亭,陳宏文,張茂山 | |
dc.subject.keyword | 組織缺血與再灌注,丙酮酸,檸檬酸合成酶,異檸檬酸脫氫酶,硫氧還蛋白還原酶, | zh_TW |
dc.subject.keyword | Ischemia/reperfusion,pyruvate,citrate synthase,NADP+-dependent isocitrate dehydrogenase,thioredoxin reductase, | en |
dc.relation.page | 44 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2012-08-08 | |
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 |
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