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研究生:許家蓁
研究生(外文):Chia-Chen Hsu
論文名稱:Bis(phenylidenebenzeneamine)-1-disulfide衍生物在生物活性上的探討
論文名稱(外文):Bioevaluation of bis(phenylidenebenzeneamine)-1-disulfide derivatives
指導教授:胡婉萍
指導教授(外文):Wan-Ping Hu
學位類別:碩士
校院名稱:高雄醫學大學
系所名稱:生物科技學系碩士班
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:2013
畢業學年度:101
語文別:中文
論文頁數:49
中文關鍵詞:黑色素瘤雙硫鍵化合物
外文關鍵詞:melanomadisulfide derivatives
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雙硫鍵在蛋白質的穩定性上佔有相當重要的地位,在抗菌及抑制特定癌細胞的研究上也有良好效果;在本篇論文中,我們得到王志鉦教授實驗室合成以雙硫鍵為主要結構的系列化合物-bis(phenylidenebenzeneamine)-1-disulfide衍生物,利用MTT assay來測定對癌細胞的毒殺效果,發現多數的化合物對不同的癌細胞都具有良好的抑制效果,但不會對正常人類纖維母細胞造成傷害;其中以B、D、L、xp-49、xp-58五種化合物對A2058以及RPMI7951這兩株黑色素瘤細胞具有較好的毒殺效果,於是選定此兩株黑色素瘤細胞作為實驗主要目標。為了由細胞週期的變化中尋找出可能的調控因子,我們利用流式細胞儀偵測雙硫鍵系列化合物對細胞週期的影響,發現這兩株黑色素瘤細胞並沒有產生顯著的細胞週期變化。由於細胞的凋亡過程,常常被聯想與粒線體的功能缺失有關,於是我們從與粒線體功能相關的細胞內活性氧化物、粒線體膜電位、細胞內ATP含量以及電子傳遞鏈中的呼吸酵素複合體次單元蛋白質表現量來做探討;結果發現在經過化合物D的作用後,A2058和RPMI7951細胞的胞內活性氧化物上升,而粒線體膜電位、細胞內ATP含量以及電子傳遞鏈中的呼吸酵素複合體次單元蛋白質表現量皆顯著下降,證明了化合物D是從造成粒線體的功能損傷來導致此兩株黑色素瘤細胞的凋亡。我們以C57BL/6小鼠作為模式物種,將小鼠黑色素瘤細胞B16打入小鼠腳掌中形成腫瘤,發現化合物D在模式物種中對腫瘤的抑制同樣具有良好的效果。綜合以上的結果,我們認為bis(phenylidenebenzeneamine)-1-disulfide衍生物可做為治療黑色素瘤的潛力藥物。

Molecules containing a disulfide moiety play a vital role in chemistry and biochemistry. In particular, disulfide-containing molecules appeared in a variety of biologically active target molecules such as anticancer activity. In the present study, we evaluated the cytotoxicity of the bis(phenylidenebenzeneamine)-1-disulfide derivatives from Dr. J. J. Wang’s lab by the MTT assay. Most of the compounds exhibited inhibitory activity on several cancer cell lines. Because five agents (B, D, L, xp-49, xp-58) exhibited a higher inhibitory activity on A2058 and RPMI7951 cells compared to other cell lines, the A2058 and RPMI7951 cells were selected as models for further studies. To verify whether cell damage might have become arrested at any cell cycle phase by bis-disulfides, the DNA content of cell nuclei was measured by flow cytometric analysis. Our results showed that no any significant phase change after bis-disulfides treatment. Because degradative changes associated with apoptosis are often preceded by the disruption of mitochondrial function, the ROS level, mitochondria membrane potential (MMP), ATP contents, and mitochondrial respiratory chain were investigated. Our data revealed that treatment of A2058 and RPMI7951 cells with compound D resulted in the ROS generation, a decrease in MMP, ATP, and proteins expression of mitochondrial respiratory chain subunits. Bis-disulfides also inhibited growth of murine melanoma B16 cells, but had no cytotoxicity to human fibroblasts. Compound D also could reduce murine melanoma size in the mouse model. Collectively, the bis(phenylidenebenzeneamine)-1-disulfide derivatives could be the promising drugs for melanoma treatment.

目錄
中文摘要……………………………………1
英文摘要……………………………………3
緒論…………………………………………4
黑色素瘤介紹………………………………………………4
雙硫鍵藥物介紹……………………………………………5
粒線體在細胞凋亡中的角色………………………………5
研究方法……………………………………7
細胞培養……………………………………………………7
細胞存活率測試 (MTT assay)……………………………8
細胞週期 (Cell cycle)偵測………………………………10
細胞內活性氧化物 (Reactive oxidative species, ROS)偵測
……………………………………………………………11
粒線體膜電位(ΔΨmt)測定………………………………13
細胞內三磷酸腺苷 (Adenosine triphosphate, ATP)偵測…14
蛋白質萃取………………………………………………15
粒線體蛋白質萃取 (Mitochondria isolation protein)……16
西方點墨法 (Western blot)………………………………17
動物腫瘤實驗……………………………………………20
結果與討論………………………………23
- Bis(phenylidenebenzeneamine)-1-disulfide衍生物對人類黑色素瘤
A2058以及RPMI7951具有較佳毒殺效果………………………21
- Bis(phenylidenebenzeneamine)-1-disulfide衍生物對A2058、
RPMI7951以及B16細胞週期的影響……………………………22
- Bis(phenylidenebenzeneamine)-1-disulfide衍生物D增加A2058、
RPMI7951和B16細胞內的活性氧化物…………………………23
- Bis(phenylidenebenzeneamine)-1-disulfide衍生物D降低A2058、
RPMI7951和B16細胞粒線體膜電位……………………………24
- Bis(phenylidenebenzeneamine)-1-disulfide衍生物降低A2058和
RPMI7951細胞內的ATP產生……………………………………24
- Bis(phenylidenebenzeneamine)-1-disulfide衍生物D降低A2058和
RPMI7951細胞粒線體內呼吸酵素複合體(Respiratory enzyme
complexes)的表現量………………………………………………25
- Bis(phenylidenebenzeneamine)-1-disulfide衍生物D抑制黑色素腫瘤
成長於動物模式中…………………………………………………26
結論………………………………………27
圖表………………………………………31
表一.Bis(phenylidenebenzeneamine)-1-disulfide衍生物取代基表格
…………………………………………………………………31
表二.化合物xp-49在不同癌細胞的毒殺效果………………………32
表三.Bis(phenylidenebenzeneamine)-1-disulfide衍生物對不同癌細胞
株的毒殺效果………………………………………………………33
表四.Bis(phenylidenebenzeneamine)-1-disulfide衍生物對不同癌細胞
株的毒殺效果………………………………………………………34
表五.Bis(phenylidenebenzeneamine)-1-disulfide衍生物對A2058、
RPMI7951和B16細胞週期影響…………………………………36
圖一.以雙硫鍵為主體所設計之衍生物主要結構……………………31
圖二.Bis(phenylidenebenzeneamine)-1-disulfide衍生物對A2058細胞
形成sub-G1之情形………………………………………………37
圖三.Bis(phenylidenebenzeneamine)-1-disulfide衍生物對RPMI7951
細胞形成sub-G1之情形…………………………………………38
圖四.Bis(phenylidenebenzeneamine)-1-disulfide衍生物對B16細胞形
成sub-G1之情形…………………………………………………39
圖五.Bis(phenylidenebenzeneamine)-1-disulfide衍生物D對A2058、
RPMI7951以及B16細胞內活性氧化物的影響…………………40
圖六.Bis(phenylidenebenzeneamine)-1-disulfide衍生物D對A2058、
RPMI7951以及B16細胞粒線體膜電位的影響…………………41
圖七.Bis(phenylidenebenzeneamine)-1-disulfide衍生物D對A2058和
RPMI7951細胞ATP含量的影響…………………………………42
圖八.Bis(phenylidenebenzeneamine)-1-disulfide衍生物D對A2058細胞
粒線體電子傳遞鏈上呼吸酵素複合體的影響…………………43
圖九.Bis(phenylidenebenzeneamine)-1-disulfide衍生物D對RPMI7951
胞粒線體電子傳遞鏈上呼吸酵素複合體的影響……………44
圖十.Bis(phenylidenebenzeneamine)-1-disulfide衍生物D對小鼠黑色素
腫瘤成長於動物模式中的影響…………………………………45
參考文獻…………………………………46

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