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研究生:王一光
研究生(外文):Yi-Kuang Wang
論文名稱:陳皮萃取物抑制內毒素對巨噬細胞與微神經膠細胞誘導發炎蛋白作用探討
論文名稱(外文):Chenpi Extract Inhibits Endotoxin Induced Inflammatory Protein Activites in Macrophages and Microglias
指導教授:何豐名何豐名引用關係翁清松翁清松引用關係
指導教授(外文):Feng-Ming HoChing-Sung Weng
學位類別:碩士
校院名稱:中原大學
系所名稱:醫學工程研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:99
中文關鍵詞:誘導型一氧化碳合成酶一氧化碳陳皮萃取物第二型環氧化酶
外文關鍵詞:nitric oxideCOX-2Chen-pi extractiNOS
相關次數:
  • 被引用被引用:2
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巨噬細胞(Macrophages)是防禦外來感染原的重要防線之一,微神經膠細胞(Microglias)則在中樞神經中有著與巨噬細胞相同的功能,它們在人體中的防禦能力佔有很重要的地位,當身體受到外來物質(細菌或其他物質)入侵或感染時,身體中的巨噬細胞與微神經膠細胞會主動因刺激而分泌許多細胞激素與一氧化氮(發炎指標物之一)來保護身體避免傷害,但如果一氧化氮分泌不足或過量,對於周遭細胞、組織、器官都將造成一定的危害。
天然中藥-陳皮經溶劑萃取出柚皮素(Naringenin)為Flavanones的結構異構物,也是一種抗氧化劑,而Flavanones對於細胞過度發炎及腫瘤的抑制,有一定的效果。
本研究首先利用柚皮素(10-200μM)對小鼠巨噬細胞與微神經膠細胞(30-100μM)之存活率及分泌一氧化氮的影響,再利用1 �慊/ml的脂多醣 ( Lipopolysaccharide, LPS )處理小鼠巨噬細胞及微神經膠細胞,使細胞受脂多醣刺激而分泌大量一氧化氮,再利用添加柚皮素的方式,抑制其發炎現象。最後利用西方墨點法證實,一氧化氮降低是因為添加柚皮素抑制了誘導型一氧化碳合成酶(iNOS, inducible nitric oxide synthase)。
實驗結果發現加入柚皮素後,細胞不會因柚皮素產生發炎反應,但細胞細胞經脂多醣處理後,會因添加柚皮素而使一氧化氮產生量降低,並且依柚皮素濃度增加使抑制能力上升。在蛋白質的結果方面,我們看到了一氧化氮合成酶(nitric oxide synthase, NOS)與第二型環氧化酶( Cyclooxygenase, COX-2 ) 濃度,會因柚皮素增加而降低。所以我們大膽推論,適量的柚皮素在正常的情況下,不會使細胞受到傷害,也不會引起細胞的發炎反應,在細胞發炎反應中,卻可以有效的抑制過度發炎的情況,經由抑制一氧化氮合成酶來降低一氧化氮的生成,也抑制第二型環氧化酶反應。
Macrophages play a key role in the immune protection system against microorganisms or tumour cells since they partake in the processing of antigens and presentation to lymphocytes, as well as in the phagocytosis and killing of microbes. In this process, high amounts of nitric oxide (NO) and reactive oxygen intermediates are generated contributing to intracellular destructive mechanisms.
While parts of our body are infected with bacteria, macrophages and microglias will secrete cytokines to protect us from being injured. However, if the system secretes too much or too little cytokine, it will cause a threat to cells, tissues, or organs.
Naringenin extract of Chen-pi of Chinese herbal medicine, a kind of antioxidant, is composed of flavanones. Furthermore, flavanones can prevent bacteria and tumors from spreading.
Our study applied naringenin (10-200μM) on cells, and it made an impact on the survival rate of Macrophages and Microglia (30-100μM). In addition, when we treated the macrophages and the microglias with LPS, it stimulated cells to secrete a large amount of nitric oxide. Besides, we treated lipopolysaccharide (LPS) on macrophages and microglias and it stimulated cells to secrete nitric oxide. Also, the study used naringenin to treat the cells and restrained them from being inflamed. According to Western Blot, nitric oxide decreased because naringenin restrained inducible nitric oxide synthase (iNOS) from developing.
In the experiment, our results indicated that the cells would not be inflamed by naringenin. However, after treating LPS stimulation, nitric oxide could be reversed by naringenin. As for the expression of proteins after LPS treatment, the expression of inducible nitric oxide synthase and cyclooxygenase (COX-2) decreased when naringenin was increased. When naringenin was added to the cells, it was able to reduce the amount of iNOS, which diminished the nitric oxide, and COX-2. Therefore, it could be concluded that the cells would not be inflamed or damaged under the proper amount of naringenin.
目錄
摘 要 I
ABSTRACT III
目錄 VI
圖目錄 X
表目錄 XIII
縮寫表 XIV
第一章 緒論 1
1-1 前言 1
1-2研究背景 3
1-3文獻回顧 5
1-4研究目的 7
第二章 理論基礎 8
2-1 中藥陳皮(Chen-Pi) 8
2-2巨噬細胞(Macrophages) 10
2-3微神經膠細胞(Microglias) 11
2-4脂多醣(Lipopolysaccharide, LPS) 12
2-5發炎(Inflammation) 15
2-5.1巨噬細胞與發炎 15
2-5.2一氧化氮(Nitric oxide, NO) 16
2-6分析方法 18
2-6.1 NO分析(Nitric Oxide Assay) 18
2-6.2 MTT分析(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) 19
2-6.3電泳(Electrophoresis, EP) 20
2-6.4西方墨點法 (Western Blot) 22
2-7 統計方法 23
第三章 實驗材料與方法 24
3-1實驗材料 24
3-1.1藥物與試劑 24
3-1.2 實驗細胞株 25
3-2細胞培養(Cell Cuture) 26
3-2.1冷凍細胞活化方法 26
3-2.2細胞繼代 26
3-2.3冷凍細胞 27
3-2.4 細胞計數方法 28
3-3藥品配置 30
3-3.1培養基配置方法 30
3-3.2 PBS配置方法 31
3-3.3 MTT溶液配置方法 31
3-3.4 Griess Reagent試劑配製(一氧化氮試劑) 32
3-3.5 Sample Buffer配置 32
3-4 實驗流程 33
3-4.1 抽取細胞蛋白Protein Extract 33
3-4.2 蛋白質定量 Protein Assay 34
3-4.3 西方墨點分析 Western Blot 35
3-4.4 細胞存活率分析MTT Assay 37
3-4.5 一氧化氮含量分析Nitric Oxide Assay 37
3-4.6 統計方法 Statistical Analysis System 37
3-4.7 圖表量化 Image Quantification 37
第四章 結果與討論 38
4-1 脂多醣與柚皮素各濃度對巨噬細胞存活率分析 38
4-2 脂多醣與柚皮素各濃度對巨噬細胞一氧化氮分析 42
4-3 細胞經脂多醣處理後加入柚皮素各濃度對巨噬細胞一氧化氮分析 45
4-4 經脂多醣處理後加入柚皮素各濃度對巨噬細胞西方墨點法分析 51
4-5 脂多醣與柚皮素各濃度對微神經膠細胞存活率分析 57
4-6 脂多醣與柚皮素各濃度對微神經膠細胞一氧化氮分析 62
4-7 經脂多醣處理後加入柚皮素各濃度對微神經膠細胞西方墨點法分析 70
第五章 結論 76
第六章 參考文獻 79


圖目錄
圖2. 1 陳皮(Chen-pi) 9
圖2. 2 脂多醣(lipopolysaccharide, LPS) 13
圖2. 3 nitric oxide assay 18
圖2. 4黃色Tetrazolium salt被切開形成紫色MTT Formazan 19
圖2. 5電泳示意圖 21
圖3. 1血球計數器 28
圖3. 2 計算示意圖 29
圖4. 1脂多醣各濃度對細胞影響之MTT吸光值 39
圖4. 2柚皮素各濃度與Vehicle組對細胞影響之MTT吸光值 40
圖4. 3柚皮素各濃度與Vehicle組對細胞影響之MTT吸光值 41
圖4. 4脂多醣各濃度使細胞所產生Nitrite之影響 43
圖4. 5脂多醣各濃度使細胞所產生Nitrite之影響) 44
圖4. 6脂多醣 0.01�慊/ml與柚皮素各濃度使細胞所產生Nitrite之影響 45
圖4. 7脂多醣 0.1�慊/ml與柚皮素各濃度使細胞所產生Nitrite之影響 46
圖4. 8脂多醣 1�慊/ml與柚皮素各濃度使細胞所產生Nitrite之影響 48
圖4. 9脂多醣 1�慊/ml與柚皮素及Vitamin C各濃度使細胞所產生Nitrite之影響 50
圖4. 10脂多醣 1�慊/ml與柚皮素各濃度使細胞所產生iNOS之影響 53
圖4. 11經Image Quant Version 5.0軟體換算脂多醣 1�慊/ml與柚皮素各濃度使細胞所產生iNOS之影響 53
圖4. 12脂多醣 1�慊/ml與柚皮素各濃度使細胞所產生第二型環氧化酶之影響 56
圖4. 13經Image Quant Version 5.0軟體換算脂多醣 1�慊/ml與柚皮素各濃度使細胞所產生第二型環氧化酶之影響 56
圖4. 14脂多醣各濃度對細胞影響之MTT吸光值 58
圖4. 15柚皮素各濃度與Vehicle組對細胞影響之MTT吸光值 60
圖4. 16柚皮素各濃度與Vehicle組對細胞影響之MTT吸光值 61
圖4. 17脂多醣各濃度使細胞所產生Nitrite之影響 63
圖4. 18脂多醣各濃度使細胞所產生Nitrite之影響 64
圖4. 19脂多醣 0.1�慊/ml與柚皮素各濃度使細胞所產生Nitrite之影響 65
圖4. 20脂多醣 1�慊/ml與柚皮素各濃度使細胞所產生Nitrite之影響 67
圖4. 21脂多醣 1�慊/ml與柚皮素及Vitamin C各濃度使細胞所產生Nitrite之影響 69
圖4. 22脂多醣 1�慊/ml與柚皮素各濃度使細胞所產生iNOS之影響 72
圖4. 23經Image Quant Version 5.0軟體換算脂多醣 1�慊/ml與柚皮素各濃度使細胞所產生iNOS之影響 72
圖4. 24脂多醣 1�慊/ml與柚皮素各濃度使細胞所產生第二型環氧化酶之影響 75
圖4. 25經Image Quant Version 5.0軟體換算脂多醣 1�慊/ml與柚皮素各濃度使細胞所產生第二型環氧化酶之影響 75


表目錄
表 1 PBS配置 31
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