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研究生:蔣宛儒
研究生(外文):Wen-Ru Jiang
論文名稱:大蒜化合物之類似物烯丙基硒半胱胺酸對於DMBA/TPA誘導皮膚腫瘤形成之影響
論文名稱(外文):The effects of Se-allylselenocysteine (ASC), an analogue of garlic compound, on DMBA/TPA-induced skin tumorigenesis
指導教授:潘敏雄潘敏雄引用關係
指導教授(外文):Min-Hsiung Pan
口試委員:王應然何元順
口試委員(外文):Ying-Jan WangYuan-Soon Ho
口試日期:2015-07-21
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:食品科技研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:66
中文關鍵詞:抗發炎烯丙基硒半胱胺酸誘導型一氧化氮合成酶環氧化酵素2DMBA/TPA誘導皮膚致癌模式
外文關鍵詞:anti-inflammationSe-allylselenocysteine (ASC)iNOSCOX-2DMBA/TPA-induced skin tumorigenesis
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烯丙基硒半胱胺酸 (Se-allylselenocysteine, ASC) 為有機硫化物之結構相似物,先前研究證實其在體外和體內實驗中能抑制乳腺癌之發生。然而,ASC對抗發炎之功效仍是未知。有越來越多的證據顯示,慢性發炎與部分腫瘤生成有很大的相關性,因此調節發炎過程之中間產物例如:誘導型一氧化氮合成酶 (Inducible nitric oxide synthase, iNOS) 和環氧化酵素2 (Cyclooxygenase-2, COX-2) 可降低癌症發生之可能性。本研究以體外細胞實驗探討ASC對於脂多醣 (Lipopolysaccharide, LPS) 誘導RAW 264.7細胞發炎之功效,進一步以ICR female mice進行7, 12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) 誘導皮膚致癌模式,探討ASC對腫瘤生成之功效。細胞實驗結果顯示,ASC在50M下抑制LPS誘導RAW 264.7的NF-B活性,進而抑制一氧化氮 (Nitric oxide ,NO) 產量和iNOS蛋白質表現量;另一方面,觀察到COX-2蛋白質和基因表現增加。在動物實驗過程中,Positive control組在第十一週時腫瘤發生率達百分之百,而ASC 5 mol/200 L實驗組則在第九週時腫瘤發生率已達百分之百;Positive control組實驗二十週結束後,每隻小鼠之腫瘤數平均約有22顆,ASC 5 mol/200 L實驗組約為35顆。小鼠之腫瘤大小分布方面,處理ASC實驗組與Positive control組比較起來有較多的趨勢。因此ASC在DMBA/TPA誘導動物致癌模式下,可能會加速乳突瘤之形成、增加腫瘤數量和腫瘤大小。同時表現ASC可促進DMBA/TPA所誘導之COX-2蛋白質表現量。綜合目前結果,推測ASC在DMBA/TPA誘導的皮膚致癌模式中,COX-2的上調對腫瘤促進扮演重要的角色,至於其詳細之作用機轉仍需進一步的探討。

Se-allylselenocysteine (ASC), an analogue of garlic compound, has been shown to inhibit mammary carcinogenesis in vivo and cell growth in vitro. However, the function of ASC on anti-inflammatory effect remains largely unknown. Therefore, we investigated whether ASC has anti-inflammatory on LPS-induced inflammation in vitro or anti-tumor promoting on 7, 12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin tumorigenesis in vivo and tried to elucidate the mechanisms involved. In in vitro study, the result showed that ASC inhibited lipopolysaccharide (LPS)-induced production of nitric oxide (NO) with decreased protein level of inducible nitric oxide synthase (iNOS) in RAW 264.7 cells. ASC also reduced nuclear factor-B (NF-B) luciferase activity. On the other hand, ASC can enhance LPS-induced COX-2 protein level and mRNA expression in RAW 264.7 cells. In in vivo study, topical application of ASC on the dorsal skin of DMBA-initiated and TPA-promoted mice could significantly accelerate skin tumorigenesis and raise tumor multiplicity than positive control group (DMBA+TPA). The number of tumors that were 1–3 mm, 3–5 mm and >5 mm in size per mouse was increased in a dose-dependent manner in the ASC-pretreated groups. Pretreatment with ASC showed significant increment on the expression of COX-2 compared with positive control group. In summary, the present results speculate that COX-2 played a crucial role in tumor-promoting effect of ASC on DMBA/TPA-induced skin cancer in mice.

謝誌 I
摘要 III
Abstract IV
目錄 V
圖表目錄 VIII
縮寫表 IX
第壹章、緒論 1
第貳章、文獻回顧 2
第一節、發炎 (Inflammation) 2
(一)、發炎反應 (Inflammatory response) 2
(二)、巨噬細胞與發炎反應 (Macrophage and inflammatory response) 3
(三)、發炎反應與腫瘤形成 (Inflammatory response and tumorigenesis) 4
第二節、發炎調控因子 (Inflammatory mediators) 5
(一)、一氧化氮 (NO) 與一氧化氮合成酶 (iNOS) 5
(二)、前列腺素 (Prostaglandins, PG) 與環氧化酵素 (COX) 6
(三)、轉錄因子NF-B 9
第三節、脂多醣 (Lipopolysaccharide, LPS) 10
(一)、脂多醣結構 (Structure of LPS) 10
(二)、LPS活化巨噬細胞之訊息傳遞路徑 11
第四節、二階段皮膚致癌模式(Two-stage skin carcinogenesis model) 13
第五節、大蒜 (Garlic) 14
(一)、大蒜簡介與其成分 14
(二)、硫和硒類似化合物在預防癌症 (Cancer prevention) 功效之比較 15
第參章、研究目的與實驗架構 17
第一節、研究動機與目的 17
第二節、實驗架構 18
第肆章、實驗材料與方法 19
第一節、實驗材料 19
(一)、儀器設備 19
(二)、藥品試劑 21
第二節、細胞實驗方法 24
(一)、Macrophage RAW 264.7細胞解凍與繼代培養 24
(二)、細胞存活率測試 (Trypan blue reagent) 25
(三)、Nitrite assay 26
(四)、Prostaglandin E2 (PGE2) 測定 26
(五)、蛋白質電泳與西方墨點法 28
(六)、RNA的抽取 33
(七)、Reverse transcriptase-polymerase chain (RT-PCR) 34
(八)、Luciferase assay 35
第三節:動物實驗:二階段致癌模式 37
(一)、動物與試劑 37
(二)、藥品配製 37
(三)、實驗方法與操作 38
(四)、實驗動物犧牲 39
第四節、統計分析 39
第伍章、實驗結果與討論 40
第一節、細胞實驗結果 40
(一)、不同濃度之ASC對小鼠巨噬細胞RAW 264.7生長之影響 40
(二)、ASC抑制LPS所誘發RAW 264.7之Nitric oxide生成 40
(三)、ASC對LPS所誘發RAW 264.7之PGE2生成量無顯著影響 41
(四)、ASC抑制LPS誘發RAW 264.7所產生的iNOS蛋白質表現量且同時促進LPS誘發RAW 264.7所產生的COX-2蛋白質表現量 41
(五)、ASC增加 LPS誘發RAW 264.7所產生之COX-2基因表現 42
(六)、ASC對LPS誘發RAW 264.7之IkB蛋白質磷酸化與降解的作用 42
(七)、ASC對LPS誘發RAW 264.7之NF-B活化作用 43
第二節、動物實驗結果 43
(一)、二階段致癌模式促癌過程中之小鼠體重變化情形 44
(二)、二階段致癌模式促癌過程中之小鼠腫瘤生長情形及腫瘤大小 44
(三)、ASC上調DMBA/ TPA誘導皮膚腫瘤的COX-2蛋白質表現量 45
第陸章、結論 47
第柒章、圖表結果 48
第捌章、參考文獻 59



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