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研究生:林儒郁
研究生(外文):LIN,RU-YU
論文名稱:表現人類重組酪胺酸酶對黑色素相關基因之研究
論文名稱(外文):Study on the Effect of Human Recombinant Tyrosinase on Melanin-Related Genes
指導教授:林智健
指導教授(外文):LIN, CHIH-CHIEN
口試委員:林智健詹錦豐陳宜嫻
口試委員(外文):LIN, CHIH-CHIENCHAN, CHIN-FENGCHEN, YI-SHYAN
口試日期:2019-07-04
學位類別:碩士
校院名稱:靜宜大學
系所名稱:化粧品科學系
學門:民生學門
學類:美容學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:82
中文關鍵詞:酪胺酸酶黑色素生成基因調控
外文關鍵詞:TyrosinaseMelanin productionGene regulation
相關次數:
  • 被引用被引用:1
  • 點閱點閱:353
  • 評分評分:
  • 下載下載:25
  • 收藏至我的研究室書目清單書目收藏:0
現今美白測試的實驗中有許多的驗證方法,在生物試驗方中大多使用細胞測試實驗,實驗中普遍所使用的細胞株大多為B16-F10小鼠黑色素瘤細胞,較少使用人類細胞,例如人類黑色素瘤細胞MeWo,因產生的黑色素含量較少,無法作為美白試驗的主要細胞株。為了希望在化粧品原料的研發過程中對細胞試驗結果有更好的可信度,因此研究其相關基因調控。我們將構築的人類重組酪氨酸酶質體(pcDNA3.1(+)-hTYR)轉染進入MeWo細胞後發現酪氨酸酶活性有顯著上升,但相對在黑色素含量試驗中黑色素沒有顯著增加。因此本研究將藉由人類重組酪氨酸酶質體共轉染進入先前本實驗室已建構好之調控黑色素生成螢光報導基因細胞中(MeWo-pMITF、MeWo-pTyr cells)進行螢光蛋白表現差異試驗,進一步探討將大量酪氨酸酶送入細胞後是否會影響相關基因的表現,進而導致黑色素無法大量產生。經由結果顯示人類重組酪氨酸酶進行轉染後具有顯著酪胺酸酶活性,但對於MITF和Tyr prmoter 並沒有產生很大,而在人類黑色素瘤細胞中可能不會產生大量的黑色素。

Nowadays, there are many verification methods for the whitening effect examination. Most of the studies were used B16-F10 mouse melanoma cells in the experiments, but less human cells, such as human melanoma cells MeWo, due to its low content of melanin, cannot be used as the main cell line for the whitening test. In order to have better reliability in the results for experiments for the development of cosmetic ingredients, we study the regulations of melanin production related genes. Previous experiments showed that the transfection of recombinant human tyrosinase expression plasmid (pcDNA3.1(+)-hTYR) into MeWo cells may found a significant increase in tyrosinase activity, but melanin content. Therefore, in this study, we co-transfected with recombinant human tyrosinase plasmid into the previously established melanin-related fluorescent reporter cells (MeWo-pMITF, MeWo-pTyr cells) to examine the fluorescence intensity differences, whether the production of large amounts of recombinant tyrosinase in human melanoma cells would affect the expression of melanin production related genes. The results showed an increasing tyrosinase activity in the transfected MeWo cells. However, recombinant tyrosinase expression was not seen to have great effects on MITF and Tyr promoter activities, also on the melanin production in human melanoma cells.
摘要I
英文摘要II
目錄III
表目錄VII
圖目錄VII
第一章 緒論1
1-2皮膚的功能3
1-3黑色素的組成和功能4
1-4黑色素生成路徑5
1-5酪氨酸酶7
1-6信號路徑調節黑色素生成9
1-7MITF信號路徑12
1-8小眼相關轉錄因子MITF13
1-9影響黑色素生成之藥品16
第二章 實驗材料與方法18
一、實驗材料18
1-1儀器18
1-2試劑19
1-3藥品19
二、人類重組酪氨酸酶之質體構築21
2-1萃取人類染色體基因21
2-2聚合酶鏈鎖反應23
2-3DNA膠體電泳分析25
2-4質體構築26
三、人類重組酪氨酸酶構築之質體DNA確認27
3-1勝任細胞的制備27
3-2轉型28
3-3質體DNA確認28
3-4萃取大量質體DNA30
四、調控黑色素生成相關基因啟動子之質體構築31
4-1質體DNA制備31
4-2 聚合酶鏈鎖反應32
4-3 DNA膠體電泳分析33
4-4質體構築34
五、調控黑色素生成螢光報導基因之質體DNA確認35
5-1 勝任細胞的制備35
5-2轉型35
5-3質體DNA確認36
六、細胞培養37
6-1 細胞株來源37
6-2冷凍細胞活化37
6-3 MeWo、MeWo-pMITF、MeWo-pTyr細胞株之基礎培養基37
6-4 B16-F10細胞株之基礎培養基38
6-5 培養基配製38
6-6 細胞培養38
6-7 細胞繼代39
6-8 冷凍保存細胞39
6-9 細胞計數40
七、實驗方法41
7-1細胞株之轉染41
7-2 螢光蛋白報導基因活性分析41
7-3黑色素含量試驗42
7-4檢品配製43
八、數據分析43
第三章 結果與討論45
一、酪氨酸酶活性分析45
二、黑色素含量試驗47
2-1人類重組酪氨酸酶表現對經過茶鹼處理的人類黑色素腫瘤細胞(MeWo)中黑色素含量之影響48
2-2人類重組酪氨酸酶表現對經過粗糠柴毒素處理的人類黑色素腫瘤細胞(MeWo)中黑色素含量之影響50
2-3人類重組酪氨酸酶表現對經過茶鹼處理的小鼠黑色素腫瘤細胞(B16F10)中黑色素含量之影響52
2-4人類重組酪氨酸酶表現對經過粗糠柴毒素處理的小鼠黑色素腫瘤細胞(B16F10)中黑色素含量之影響54
三、螢光蛋白報導基因活性分析56
3-1人類重組酪氨酸酶表現對MeWo-pMITF細胞中MITF啟動子活性之影響57
3-2人類重組酪氨酸酶表現對經過茶鹼處理之MeWo-pMITF細胞中MITF啟動子活性之影響59
3-3人類重組酪氨酸酶表現對經過粗糠柴毒素處理MeWo-pMITF細胞中MITF啟動子活性之影響62
3-4人類重組酪氨酸酶表現對經過茶鹼處理MeWo-pTyr細胞Tyr啟動子活性之影響65
3-5人類重組酪氨酸酶表現對經過粗糠柴毒素處理MeWo-pTyr細胞Tyr啟動子活性之影響68
第四章 結論71
第五章 參考資料74

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