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研究生:林昭呈
研究生(外文):Zhao-Cheng Ling
論文名稱:鹼性抗生胜肽Indolicidin及其類似物對於人類皮膚細胞及人類表皮皮膚癌細胞之影響
論文名稱(外文):Biological Activities of Indolicidin and Its’ Analogues on Fibroblast, Keratinocyte and Human Epidermoid Carcinoma
指導教授:謝瑞香
指導教授(外文):Jui-Hsiang Hsieh
學位類別:碩士
校院名稱:中原大學
系所名稱:生物醫學工程研究所
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:2011
畢業學年度:99
語文別:中文
論文頁數:76
中文關鍵詞:抗癌活性鹼性抗生胜肽Indolicidin
外文關鍵詞:activity to againstcationic antimicrobial peptideIndolicidin
相關次數:
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許多文獻指出,從牛的嗜中性白血球中所分離出來的鹼性抗生胜
肽Indolicidin 具廣效抗菌性,可對抗真菌、細菌或病毒。越來越多研
究發現一些鹼性抗生胜肽其實也存在對癌細胞的抗癌活性,因此本實
驗想探討Indolicidin 及其類似物IL-K7、IL-F89 及IL-K7F89 是否會
對人類表皮膚癌細胞產生抗癌活性或對正常皮膚細胞存在細胞毒
性。
實驗希望找出適合的濃度與胜肽,對皮膚癌細胞具高抗癌活性並
且對正常皮膚細胞擁有低細胞毒性,所以本實驗選用三種濃度:60μM、
30μM 和10μM 來進行實驗,並對利用PI 染色及TUNEL assay 來觀察
細胞受胜肽作用後是經由凋亡或是壞死而導致死亡。
由實驗結果得知10μM 的IL-K7F89 有高抗癌細胞活性能抑制人
類表皮皮膚癌細胞的生長,只對角質細胞及纖維母細胞產生些微的細
胞毒性。由PI 染色和TUNEL assay 結果得知,IL 及其類似物會造成
細胞壞死並非凋亡。之前研究也證實IL-K7F89 具有比IL 高的抗菌活
性也具備比IL 低的溶血活性,結合這些優點或許未來可將其應用在
治療表皮皮膚癌細胞上。


Many literatures indicated that indolicidin(IL), one of cationic
antimicrobial peptides(CAPs) isolated from the cytoplasmic granules of
bovine neutrophils, has broad-spectrum antimicrobial activity. It can
inhibit the growth of bacteria, fungi and even viruses. A growing number
of studies have shown that some of cationic antimicrobial peptides may
own cytotoxic activity against cancer cells. We, therefore, try to
investigate the cytotoxic activity of IL and its analogues against human
epithelial carcinoma A431and normal human skin cells, HS68 and
CRL2309.
We vary the concentration of IL and its analogues and investigate
the relationship between peptide concentration and cell toxicity. The
suitable concentrations may be found to have high activity against human
epithelial carcinoma but low cytotoxicity toward normal fibroblasts and
keratinocytes. Three concentrations were tested: 60μM, 30μM and 10μM.
The result showed that one of the IL analogue, IL-K7F89, has the highest
anticancer activity to A431 and the lowest cytotoxic activities toward
HS68 and CRL2309 at 10μM. By using PI stain and TUNEL assay, we
also found that indolicidin and its analogues kill cells by direct necrosis
but not inducing apoptosis. In addition, previous study had indicated that
IL-K7F89 has higher antimicrobial but lower hemolytic activity than IL
does. Our study further indicated that the IL analogue, IL-K7F89, will
also be a good candidate for cancer treatment.
中文摘要 ...................................................................................................... I
英文摘要 .................................................................................................... II
致謝 ........................................................................................................... III
目錄 ........................................................................................................... IV
圖目錄 ..................................................................................................... VII
表目錄 ........................................................................................................ X
第一章 緒論............................................................................................. 1
第二章 文獻回顧 .................................................................................... 3
2-1 鹼性抗生胜肽 .................................................................................. 3
2-1-1 鹼性抗生胜肽的特徵 ........................................................... 3
2-1-2 鹼性抗生胜肽的抗生機制 ................................................... 3
2-1-3 鹼性抗生胜肽的抗癌活性 ................................................. 10
2-2 Indolicidin 及其類似物 ................................................................. 13
2-2-1 Indolicidin 的特徵 ............................................................... 13
2-2-2 Indolicidin 的抗生機制 ....................................................... 14
2-2-3 Indolicidin 的類似物 ........................................................... 16
第三章 材料與方法 .............................................................................. 19
3-1 實驗設備 ........................................................................................ 19
3-2 實驗藥品 ........................................................................................ 21
3-3 藥品製備 ........................................................................................ 24
3-3-1 PBS 製備 ............................................................................. 24
3-3-2 Trypsin inhibitor 溶液製備 ................................................. 24
3-3-3 膠原蛋白溶液製備 ............................................................. 25
3-3-4 Indolicidin 及其類似物溶液製備 ....................................... 25
3-3-5 DMEM 培養基製備 ............................................................ 26
3-3-6 PI 染劑製備 ......................................................................... 26
3-3-7 Fixation solution 製備 ......................................................... 27
3-3-8 Blocking solution 製備 ........................................................ 27
3-4 細胞培養 ........................................................................................ 28
3-4-1 人類皮膚纖維母細胞 ......................................................... 28
3-4-2 人類角質細胞 ..................................................................... 30
3-4-3 人類表皮皮膚癌細胞 ......................................................... 32
3-5 實驗架構 ........................................................................................ 34
3-6 實驗方法 ........................................................................................ 35
3-6-1 生長曲線 ............................................................................. 35
3-6-2 觀察細胞型態 ...................................................................... 35
3-6-3 細胞壞死測定 ..................................................................... 36
3-6-4 細胞凋亡時DNA 片段化測定 ........................................... 37
第四章 結果與討論 .............................................................................. 39
4-1 皮膚細胞受Indolicidin 及其類似物作用後的生長曲線 ............ 39
4-1-1 A431 與IL 及其類似物作用後之生長曲線....................... 39
4-1-2 Keratinocyte 與IL 及其類似物作用後之生長曲線 ........... 42
4-1-3 Fibroblast 與IL 及其類似物作用後之生長曲線 ............... 45
4-2 皮膚細胞受Indolicidin 及其類似物作用後的細胞型態 ............. 53
4-2-1 A431 與IL 及其類似物作用後之細胞型態....................... 53
4-2-2 Keratinocyte 與IL 及其類似物作用後之細胞型態 ........... 54
4-2-3 Fibroblast 與IL 及其類似物作用後之細胞型態 ............... 55
4-3 Indolicidin 及其類似物造成皮膚細胞的凋亡或壞死 .................. 56
4-3-1 A431 與IL 及其類似物作用後之PI 染色 ......................... 56
4-3-2 A431 與IL 及其類似物作用後之TUNEL assay ............... 58
4-3-3 Keratinocyte 與IL 及其類似物作用後之PI 染色 ............. 60
4-3-4 Keratinocyte 與IL 及其類似物作用後之TUNEL assay ... 62
4-3-5 Fibroblast 與IL 及其類似物作用後之PI 染色 .................. 64
4-3-6 Fibroblast 與IL 及其類似物作用後之TUNEL assay ....... 66
第五章 結論........................................................................................... 71
參考文獻 ................................................................................................... 72
圖目錄
圖2-1 磷脂質構造 ................................................................................. 5
圖2-2 微生物與人類紅血球細胞膜的組成成份比率圖 .................... 6
圖2-3 CAPs 吸附在磷脂質雙層膜表面 ............................................. 6
圖2-4 Barrel Stave ................................................................................. 7
圖2-5 Toroidal Pore ............................................................................... 7
圖2-6 Carpet modle ............................................................................... 7
圖2-7 Molecular electroporation model ................................................ 8
圖2-8 Sinking raft mode ........................................................................ 8
圖2-9 CAPs 在細胞內外的作用機制 ................................................. 9
圖2-10 不同濃度Indolicidin 及其類似物之溶血百分比 ................. 17
圖3-1 顯微鏡下的人類皮膚包皮纖維母細胞 .................................. 29
圖3-2 顯微鏡下的人類角質細胞 ....................................................... 31
圖3-3 顯微鏡下的人類表皮皮膚癌細胞 .......................................... 33
圖3-4 實驗組別圖 ............................................................................... 34
圖4-1 60μM IL 及其類似物對A431 作用後之生長曲線 ................ 40
圖4-2 30μM IL 及其類似物對A431 作用後之生長曲線 ................ 41
圖4-3 10μM IL 及其類似物對A431 作用後之生長曲線 ................ 42
圖4-4 60μM IL 及其類似物對Keratinocyte 作用後之生長曲線 .... 43
圖4-5 30μM IL 及其類似物對Keratinocyte 作用後之生長曲線 .... 44
圖4-6 10μM IL 及其類似物對Keratinocyte 作用後之生長曲線 .... 44
圖4-7 60μM IL 及其類似物對Fibroblast 作用後之生長曲線 ......... 45
圖4-8 30μM IL 及其類似物對Fibroblast 作用後之生長曲線 ......... 46
圖4-9 10μM IL 及其類似物對Fibroblast1 作用後之生長曲線 ....... 47
圖4-10 A431 與IL 及其類似物作用後兩天之存活率 ..................... 48
圖4-11 Keratinocyte 與IL 及其類似物作用後兩天之存活率 ......... 48
圖4-12 Fibroblast 與IL 及其類似物作用後兩天之存活率 ............. 49
圖4-13 60μM 的IL 及其類似物對皮膚細胞作用後兩天之細胞數量
................................................................................................. 50
圖4-14 30μM 的IL 及其類似物對皮膚細胞作用後兩天之細胞數量
................................................................................................. 50
圖4-15 10μM 的IL 及其類似物對皮膚細胞作用後兩天之細胞數量
................................................................................................. 51
圖4-16 A431 與IL 及其類似物作用後兩天之細胞型態 ................. 53
圖4-17 Keratinocyte 與IL 及其類似物作用後兩天之細胞型態 ..... 54
圖4-18 Fibroblast 與IL 及其類似物作用後兩天之細胞型態 ......... 55
圖4-19(A) A431 與IL 及其類似物作用後6 小時之PI 染色圖 ..... 56
圖4-19(B) A431 與IL 及其類似物作用後6 小時之PI 染色圖 ...... 57
圖4-20(A) A431 與IL 及其類似物作用後6 小時之TUNEL assay 58
圖4-20(B) A431 與IL 及其類似物作用後6 小時之TUNEL assay 59
圖4-21(A) Keratinocyte 與IL 及其類似物作用6 小時後之PI 染色圖
........................................................................................... 60
圖4-21(B) Keratinocyte 與IL 及其類似物作用6 小時後之PI 染色圖
........................................................................................... 61
圖4-22(A) Keratinocyte 與IL 及其類似物作用6 小時後之TUNEL
assay .................................................................................. 62
圖4-22(B) Keratinocyte 與IL 及其類似物作用6 小時後之TUNEL
assay .................................................................................. 63
圖4-23(A) Fibroblast 與IL 及其類似物作用後6 小時之PI 染色圖
........................................................................................... 64
圖4-23(B) Fibroblast 與IL 及其類似物作用後6 小時之PI 染色圖
........................................................................................... 65
圖4-24(A) Fibroblast 與IL 及其類似物作用後6 小時之TUNEL assay
........................................................................................... 66
圖4-24(B) Fibroblast 與IL 及其類似物作用後6 小時之TUNEL assay
........................................................................................... 67
圖4-25 A431 癌細胞PI 染色定量圖 ................................................. 69
圖4-26 CRL2309 癌細胞PI 染色定量圖 .......................................... 70
圖4-27 HS68 癌細胞PI 染色定量圖 ................................................. 70
表目錄
表2-1 具抗癌活性的鹼性抗生胜肽 ................................................... 12
表2-2 Indolicidin 的廣效抗菌活性 .................................................... 13
表2-3 Indolicidin 及其類似物序列、分子量和生理PH 值範圍內所
帶電荷數 ................................................................................... 18
表3-1 PBS 成份 ................................................................................... 24
表3-2 人類皮膚纖維母細胞資料表 ................................................... 28
表3-3 人類角質細胞資料表 ............................................................... 30
表3-4 人類表皮皮膚癌細胞資料表 ................................................... 32
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