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研究生:黃淨修
研究生(外文):Jing-ShiouHaung
論文名稱:探討巨噬細胞在IL-1β 和 TGF-β共同作用下CEBPD與其交互作用蛋白質體研究
論文名稱(外文):Investigate the interactome of macrophage CCAAT/enhancer binding protein delta in cotreatment of IL-1β and TGF-β
指導教授:王育民王育民引用關係
指導教授(外文):Ju-Ming Wang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物資訊與訊息傳遞研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:58
中文關鍵詞:CEBPDBIAcore蛋白質體蛋白質交互作用
外文關鍵詞:CEBPDBIAcoreproteomeprotein interaction
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流行病學研究指出慢性發炎會導致許多退化性疾病,例如:神經退化、動脈粥狀硬化、第二型糖尿病、類風溼性關節炎,甚至促進癌細胞形成。經數十年研究,發現在不同疾病中發炎反應的分子機轉是共通。CEBPD 的活化在許多發炎關疾病均可看見,但是其在細胞與病理進程中所扮演的角色仍有許多未知。於此,我們對於巨噬細胞受到IL-1β及TGF-β刺激後的 CEBPD 與其蛋白質交互作用的情況感到興趣。因此我們首先純化 CEBPD 重組蛋白,並結合 BIAcore 與 LC-MS/MS 兩項儀器,作為本研究中探討 CEBPD 交互作用蛋白質體研究的工具。另外,已知 SMAD3/4 為 TGF-β的下游反應因子,其可與CEBPD結合,並在不同細胞中調控 PPARG2 , COX-2與MCP-1基因轉錄。因此我們想在巨噬細胞中檢測CEBPD是否也能與 SMAD3/4 進行交互作用,並影響 PPARG2 , COX-2 與 MCP-1 報導基因的轉錄活性。
Epidemiologic studies suggest that chronic inflammation in aging is related to age-associated disorders, such as neurodegeneration, atherosclerosis, type II diabetes and RA, and enhanced risk of tumor formation. Over the past few decades, scientists have realized that the process of inflammation is virtually the same in different disease states. The activation of inflammatory cytokine-inducible transcription factor CCAAT/enhancer binding protein (CEBPD) has been observed in many inflammatory diseases states. However, the details of the molecular of CEBPD action in cells and pathogenesis remain largely uninvestigated. Herein, we are interested and trying to identify the interactome of CEBPD responding to treatments of IL-1β , TGF-β and IL-1β/TGF-β in macrophage by using a BIAcore/LC-MS/MS strategy. Following this idea, we purified recombinant CEBPD protein and confirmed the functional bindings of this recombinant protein or oligonucleotide-bound CEBPD on the chip. In addition, SMAD3/4, TGF-β responsive factors, were reported to interact with CEBPD and contributes to transcriptional activations of PPARG2 , COX-2 and MCP-1 genes indifferent cell types. We therefore test the bindings of CEBPD and SMAD3/4 and their consequent effect on PPARG2 , COX-2 and MCP-1 reporters in macrophages.
緒論...........................................................................................................................1
1. C/EBP家族成員CEBPD..................................................................................1
1-1 CCAAT/enhancer binding protein (C/EBPs) family...........................................1
1-2 CEBPD參與之生理功能與分子調控機制.......................................................2
2. TGF-β 於巨噬細胞中參與生理功能.............................................................3
3. 蛋白質體學的發展與應用................................................................................3
4. 生物表面共振系統與質譜儀之合併發展........................................................6
5. 研究動機............................................................................................................8
實驗材料與方法 .....................................................................................................9
A 細胞株培養.....................................................................................................9
B 給藥方式.........................................................................................................9
B-1 藥物配製與儲存..............................................................................................9
B-2 給藥方法..........................................................................................................9
C CEBPD 重組蛋白純化..................................................................................9
C-1 IPTG誘導蛋白質表現....................................................................................9
C-2 破菌與液相層析分析法 (Fast Performance Liquid Chromatography).........10
C-3 純化後 CEBPD 蛋白質表現染色.................................................................11
C-4 純化 CEBPD 蛋白濃縮與去鹽.....................................................................11
D 核蛋白萃取 (Nuclear proteins extraction).....................................................11
E DNA親和力沉澱分析法 (DNA affinity precipitation analysis ; DAPA) .....12
F BIAcore®生物分子交互作用分析技術 (Biomolecular interaction analysis)13
F-1 純化後的CEBPD蛋白質固定化酸鹼值測試................................................13
F-2 晶片中純化的CEBPD 蛋白質固定化..........................................................13
F-3 晶片中生物素標定之DNA-CEBPD 蛋白質固化定....................................14
F-4 晶片中純化的CEBPD蛋白質活性再生及CEBPD結合之特異性蛋白回收………………………………………………...............................................14
G 分析細胞mRNA表現.................................................................................. 15
G-1 全RNA (total RNA) 萃取 ........................................................................15
G-2 反轉錄反應....................................................................................................16
G-3 聚合脢連鎖反應...............................................................................................16
H 分析細胞蛋白質量 .........................................................................................17
H-1 蛋白質定量.......................................................................................................17
H-2 SDS-PAGE 膠體電泳製作 ............................................................................17
H-3 西方點墨法、半乾式轉漬法……………………………...............................17
H-4 化學冷光呈色分析...................................................................................................18
I 分析蛋白質間交互作用力....................................................................................19
I-1 共免疫沉澱法 (Co-immunoprecipitation) .......................................................19
J 質體構築與轉殖...............................................................................................19
J-1 質體轉殖 (Transient transfection)...................................................................20
K 細胞轉錄活性檢測 ( Luciferase reporter assay).............................................20
K-1 暫時性質體轉染 (Transient transfection).........................................................20
K-2 報導基因分析法 (Luciferase reporter assay)....................................................21
L 統計分析方法 (Statistical analysis) .................................................................21
實驗結果......................................................................................................................22
純化CEBPD之重組蛋白...........................................................................................22
純化後之CEBPD重組蛋白於晶片上進行固定化....................................................22
TGF-β和IL-1β共同刺激後能與CEBPD蛋白產生交互作用的核蛋白及蛋白回收................................................................................................................................24
CEBPD特異性結合蛋白身分鑑定..........................................................................25
TGF-β和IL-1β調控巨噬細胞內MCP-1,COX-2及PPARG2基因表達調控....30
SMAD3/4與CEBPD的蛋白質交互作用................................................................30
實驗討論 ...................................................................................................................32
CEBPD蛋白純化方式優化.......................................................................................32
CEBPD在晶片固定化條件及蛋白回收條件的優化...............................................32
CEBPD特異性結合蛋白探討...................................................................................34
TGF-β和IL-1β對巨噬細胞內MCP-1,COX-2及PPARG2轉錄調控途徑之探討……………………………………………………………………………………..36
Interactomes資料庫建立與未來之應用.....................................................................37
參考文獻.....................................................................................................................38

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