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研究生:許瓊月
研究生(外文):Chiung-Yueh Hsu
論文名稱:利用蛋白質體學研究pycnogenol(松之萃)對RAW264.7細胞整體基因表現之影響
論文名稱(外文):The investigation of the effect of pycnogenol on the global gene expression of the RAW 264.7 cells
指導教授:吳定峰吳定峰引用關係
指導教授(外文):Ting-Feng Wu
學位類別:碩士
校院名稱:南台科技大學
系所名稱:生物科技系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
中文關鍵詞:松之萃蛋白質體學
外文關鍵詞:pycnogenol
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Pycnogenol (PYC) 是由生長在法國南部沿海海岸森林中的濱海松(Pinus pinaster solander in aiton)樹皮經由熱水萃取出來,此萃取物富含多種天然物質,其中最主要含大量的類黃酮 (bioflavenoid),如花青素 (proanthocyanidin)及兒茶素(catechin) 等。
現今對pycnogenol的研究主要著重在在臨床上的應用,包括皮膚病理、心臟病、免疫系統、癌症、阿茲海默症、高膽固醇、糖尿病、關節炎、抗氧化等的研究 ,而對於影響基因表現方面的文獻較少,因此在本論文中我們使用蛋白質體學的技術來研究 pycnogenol 對於 RAW 264.7 細胞株的基因表現影響。
我們使用高解析度的二維電泳膠建立未經過 pycnogenol 處理和經過100 mg/ml pycnogenol 處理24小時的RAW 264.7細胞的二維電泳圖譜,結果在未經過處理的細胞中可偵測到713個蛋白質點,而在經過處理的細胞中有725個蛋白質點,透過 PDQUEST 軟體後比對找到4個統計上有意義並超過2倍以上表達增加的蛋白質點。利用高效能液相層析儀-電噴灑游離法-串聯式質譜儀 (HPLC-ESI/MS/MS) 我們鑑定出這些差異性的蛋白質點分別為 mouse cathepsin D、mouse proteasome a subunit type 1、annexin IV 及 keratinocyte lipid-binding protein,以西方墨點法進一確認這些蛋白質的表達是有差異的。
Pycnogenol (PYC) , whose major components are bioflavonoids, such us oligomeric proanthocyanidins and catechin is extracted with hot water from the bark of French maritime pink (Pinus pinaster solander in aiton) along the coast in the southern French.
The studies on pycnogenol mainly focused on the clinical application in the past years, including treatment for skin pathology、cardiopathy、immune system、cancer、Alzheimer’s disease、high cholesterol、diabetes、arthritis and free radical scavenger etc. However, the references regarding the effect of pycnogenol on the gene expression are very few. Therefore, in this thesis we utilized the proteomics to examine the effect of pycnogenol on the genome-wide gene expression of RAW 264.7 cells.
We used two-dimensional (2-D) gels with high resolution to establish the pH 4-7 11 —cm proteome maps of RAW 264.7 cells either non-treated or with 100 mg/ml pycnogenol for 24 hours. Using PDQUEST software, we detected 713 points on the gels of PYC-treated and 725 points on the non-treated RAW 264.7 cells. The proteome maps of non-treated and treated RAW 264.7 cells were compared with the PDQUEST software. Four statistically significant spots with two-fold or above in the amount proteins were detected. These differentially expressed protein spots were identified are cathepsin D、proteasome a subunit type 1、annexin IV and keratinocyte lipid-binding protein respectively using electrospray ionization mass spectrometry (ESI/MS/MS). These differentially expressed proteins were confirmed by the Western blot .
中文摘要…………………………………………………………………i
英文摘要…………………………………………………………………ii
圖目錄……………………………………………………………………v
表目錄……………………………………………………………………vi
第一章 前言…………………………………………………………… 1
第二章 文獻探討……………………………………………………… 3
2-1 Pycnogenol的背景介紹………………………………………3
2-2 Pycnogenol之成份……………………………………………4
2-3 Pycnogenol的抗氧化能力……………………………………4
2-4 Pycnogenol的臨床應用………………………………………6
2-5 Pycnogenol對巨噬細胞活性的調控…………………………8
2-6 Pycnogenol對細胞基因表達的影響…………………………9
2-7 蛋白質體學……………………………………………………9
2-7.1 蛋白質二維電泳………………………………………………10
2-7.2 蛋白質鑑定技術………………………………………………11
2-7.2.1 Edman sequencing……………………………………………11
2-7.2.2 蛋白質質譜鑑定………………………………………………11
第三章 材料與方法 ……………………………………………………14
3-1 Pycnogenol的來源………………………………………… 14
3-2 處理Pycnogenol 不同濃度24小時對於 TNF-α的分泌…… 14
3-3 處理Pycnogenol 不同時間點對於 TNF-α的分泌………… 14
3-4 Pycnogenol對細胞的毒性試驗………………………………15
3-5 細胞蛋白質的萃取……………………………………………15
3-6 蛋白質定量……………………………………………………16
3-7 蛋白質等電點聚焦電泳法 (isoelectric focusing, IEF)16
3-8 SDS-PAGE電泳…………………………………………………17
3-9 銀染(silver staining)…………………………………… 17
3-10 軟體比對… ………………………………………………… 18
3-11 膠內蛋白質水解………………………………………………18
3-12 電灑游離式串連質譜儀鑑定- (ESI/MS/MS)……………… 19
3-13 西方墨點法 (Western blotting) …………………………19
第四章 結果…………………………………………………………… 21
4-1 RAW264.7細胞株二維電泳條件的探討………………………21
4-2 受pycnogenol影響之RAW264.7細胞的二維電泳圖譜………21
4-3 受pycnogenol影響而表現差異的蛋白質點…………………22
4-4 表現差異蛋白質點身份之確認………………………………23
4-5 西方墨點法……………………………………………………24
4-6 Cathepsin D蛋白質點動力學分析………………………… 24
第五章 討論……………………………………………………………26
參考文獻…………………………………………………………………29
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