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研究生:范維貞
論文名稱:以HMGB1作為新穎的疾病診斷平台之標靶
論文名稱(外文):Development of novel diagnostic platform targeted to HMGB1
指導教授:金立德
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:微生物免疫與生物藥學系研究所
學門:生命科學學門
學類:其他生命科學學類
論文種類:學術論文
畢業學年度:103
語文別:中文
中文關鍵詞:高遷移性蛋白B1中國倉鼠卵巢細胞Flp-In
外文關鍵詞:HMGB1CHOFlp-In
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高遷移性蛋白B1 (High mobility group box 1 protein, HMGB1),在30年前就已經被發現,為非histone結構性蛋白,能與DNA結合,其分子量大約30 kD的蛋白質。HMGB1廣泛存在於真核細胞中,是一種胞內蛋白,在哺乳類動物具有高度的遺傳保留性。HMGB1結構由215個多數帶有多個電荷的胺基酸所組成,其結構具有兩個DNA binding domains,分別為A box domain及B box domain,並且含有一段帶有負電的 C- terminal tail。HMGB1在1999年被發現是一種晚期調節細胞激素(late mediator cytokines),但對於HMGB1所造成的發炎反應,具有不同的調控作用。
A box domain可以利用競爭方式結合HMGB1,進而抑制HMGB1及B box domain造成的發炎現象;B box domain卻是與HMGB1相同,具有促進發炎的效果。當單核球細胞及巨噬細胞被活化時,HMGB1會從胞內釋放或分泌出來。HMGB1會藉由兩種路徑釋放到細胞外:(1) 藉由IL-1、TNF-或是LPS刺激單核球細胞及巨噬細胞,使單核球細胞及巨噬細胞分泌出HMGB1,此路徑稱為主動釋放。(2) 當細胞壞死時,原本在細胞核內的HMGB1則會被表現釋放到細胞外,影響周圍的細胞產生發炎反應,此路徑稱為被動釋放。當HMGB1在人體內濃度過高時,則會對人體造成許多疾病,最終引發敗血症而死亡。本研究目的以人為主體,採用人類周邊血液細胞,刺激細胞產生源自於人的HMGB1,並將HMGB1以分生的方式進行永續生產,藉以發展出針對HMGB1的疾病檢測平台的開發。結果顯示我們不僅從人血中偵測出HMGB1序列,並與NCBI Blast進行比對,發現與人類HMGB1序列吻合。並以Flp-InTM-CHO建構出一套HMGB1蛋白的生產系統,可用於發展新的疾病試劑診斷平台的開發。
目錄 I
表目錄 V
圖目錄 VI
摘要 VII
Abstract IX
謝誌 XI
第一章、緒論 1
1.1 高遷移率族蛋白 1
1.1.1 HMGB家族 1
1.1.2 HMGB1的結構及功能 2
1.2 HMGB1為發炎介質 4
1.3 HMGB1與疾病之關聯 7
1.3.1 類風溼性關節炎 7
1.3.2 敗血症與內毒素血症 7
1.3.3 系統性紅斑性狼瘡 8
1.3.4 癌症 8
1.3.5 糖尿病 9
1.3.6 中風 9
1.3.7 肺動脈高壓 10
1.4 針對HMGB1的治療 11
1.4.1 抗HMGB1 之中和抗體 11
1.4.2 重組HMGB1 A box 片段 11
1.4.3 阻斷RAGE之訊號 12
1.4.4 兒茶素沒食子酸 12
1.4.5 丙酮酸乙酯 12
1.4.5 榭皮素 13
1.5 研究動機與目的 14
第二章、材料與方法 15
2.1 人類血球分離及刺激 15
2.1.1 人類周邊單核球細胞分離及刺激 15
2.1.2 人類白血球分離及刺激 17
2.2 抗原基因之分生表現 19
2.2.1 細胞之RNA抽取 19
2.2.2 反轉錄聚合酶連鎖反應 20
2.2.3 聚合酶連鎖反應 21
2.2.4 PCR 產物純化法 23
2.2.5 TA cloning 24
2.2.6 化學法轉形作用 26
2.2.7 電穿孔轉形作用 28
2.2.8 聚合酶連鎖反應確認 30
2.2.9 質體抽取 31
2.2.10 限制酶酵素酶切純化 32
2.2.11 膠體純化 33
2.2.12 載體置換 35
2.2.13 細胞轉染 37
2.3 HMGB1於細胞表現蛋白 40
2.3.1 中國倉鼠卵巢細胞培養 40
2.3.2 Bradford蛋白質測定法 40
2.3.3 Dot Blot試驗 41
2.4 石英壓電晶體微天平分子交互作用分析 43
2.4.1 抗體與抗原親和力之偵測 43
2.4.2 抗原決定位置的確認 45
第三章、結果 48
3.1 內毒素刺激人類PBMC及PMN產生HMGB1 48
3.2 中國倉鼠卵巢蛋白質表現 49
3.3 HMGB1抗體親和力之測定 50
3.4 Dot blot試驗結果 51
第四章、討論 52
參考文獻 59
附件一 倫理審查委員會審查文件 76
附件二 實驗流程 77
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