抗癌胜肽DI抑制人類大腸癌細胞株SW480增生並誘導細胞凋亡作用的探討

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研究生：	林芳伊 Lin, Fang-Yi
論文名稱：	抗癌胜肽DI抑制人類大腸癌細胞株SW480增生並誘導細胞凋亡作用的探討 Anticancer peptide DI inhibits the proliferation and induces apoptosis of human colorectal cancer cell line SW480
指導教授：	林志侯 Lin, Thy-Hou
口試委員:	高茂傑 Kao, Mou-Chieh 張晃猷 Chang, Hwan-You
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2017
畢業學年度：	106
語文別：	英文
論文頁數：	50
中文關鍵詞：	乾酪乳桿菌、抗癌胜肽、結腸直腸腺癌、細胞凋亡、DNA 裂解
外文關鍵詞：	Lactobacillus casei, Anticancer peptide, Colorectal adenocarcinoma, Apoptosis, DNA fragmentation
相關次數：	點閱：3 下載：0
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癌症是全球主要的公共衛生問題，而傳統治療癌症的方法具有一些有害的副作用，然而幸運的是，許多研究發現抗菌胜肽除了具有抗菌能力外，也具有促使癌細胞死亡的能力，因此這些發現使得抗菌胜肽相關的研究逐漸受到重視。近年來，本實驗室致力於原生益菌及其衍生物的相關研究，自Lactobacillus casei group資料庫中挑選出四種具有抗癌潛力之抗菌胜肽(DI, DII, EI and EII)，在先前的實驗中發現DI胜肽具有最顯著的抑制人類大腸癌細胞株SW480的能力，因此，在本篇論文中，DI胜肽為主要的實驗材料。在細胞毒性測試中，SW480細胞在DI 加藥24小時後的IC50 = 28.04 μg/mL (4.67 μM)，並發現在相同加藥時間下，其毒殺能力約是化療藥物5-氟尿嘧啶的540倍。另外，透過流式細胞儀、共軛焦顯微鏡、西方點墨法和末端脫氧核苷酸轉移酶脫氧尿苷三磷酸切口末端標記，針對DI胜肽對於SW480細胞加藥後的細胞型態、膜的通透性以及誘導的細胞死亡路徑這三個層面觀察細胞的變化。整體結果顯示DI胜肽不會造成細胞的細胞膜受損，並可能透過細胞凋亡路徑促使細胞死亡，但詳細的死亡機制仍有待日後的研究。

Cancer is a major public health problem in the world. Traditional methods of cancer treatment have some deleterious side effects on normal cells. Fortunately, some studies indicated that some antimicrobial peptides not only have been discovered with antimicrobial activity but also with anticancer activity. Therefore, recent research has paid increasing attention to antimicrobial peptides. In recent years, our laboratory has been working on antimicrobial peptide that produced by lactic acid bacteria and found four potential anticancer peptides (DI, DII, EI and EII) form Lactobacillus casei group database. As a result of previous experiments found that the survival rate of human colorectal cancer cell lines SW480 dramatically decreasing with the concentration of DI peptide increasing. In this thesis, DI peptide was the experiment material. In cell cytotoxicity test, the IC50 value of DI peptide was 28.04 μg/mL (4.67 μM) after 24 hours treatment and it had the five hundred and forty times inhibitory effect than chemotherapy drugs (5-fluorouracil) on SW480 cell during the same time. On the other hand, SW480 cell changes after treated with DI peptide were observed in three aspects：cellular morphology, membrane permeability and possible cell death pathway by flow cytometer, western blot, laser scanning confocal microscopy and terminal transferase mediated DNA nick end labelling (TUNEL) assay. The results indicated that the cell membrane remains intact after DI peptide treatment and the concerning cell death pathway may be apoptosis. However, the underlying mechanism is not yet clear, and is still further investigation.

中文摘要    ii
English Abstract    iii
Acknowledgement    iv
Content    v
1.    Introduction    1
2.    Materials and Methods    9
2.1    Cell culture    9
2.2    Cell counting    9
2.3    Peptides    10
2.4    Protein quantification    10
2.5    Freezing and thawing cultured cells    10
2.6    Circular dichroism spectroscopy    11
2.7    Cell proliferation assay    12
2.8    Flow cytometric analysis    12
2.9    Protein electrophoresis    13
2.10    Western blot analysis    14
2.11    TUNEL assay    15
2.12    Laser scanning confocal microscopy    16
2.13    Inhibition of apoptosis by pan-caspase inhibitor    17
2.14    Statistical analysis    17
3.    Results    18
3.1    The solubility of DI peptide    18
3.2    Secondary structure of DI peptide    18
3.3    Cytotoxic activities of DI peptide in SW480 cell    19
3.4    Compared the cytotoxic activity between DI peptide and 5-FU    19
3.5    Apoptosis and membrane permability detection by flow cytometric analysis    20
3.6    Effect of caspase inhibitor on DI peptide induced apoptosis    21
3.7    Expression of apoptotic markers in SW480 cells after DI peptide treatment    21
3.8    Detection of DNA strand breaks after DI peptide treatment    22
3.9    Location of DI peptide and cellular morphology by laser scanning confocal microscopy    23
4.    Discussion    25
5.    Tables and Figures    29
Figure 1. Secondary structure analysis of DI peptide    29
Figure 2. Cytotoxicity test of DI peptide    30
Figure 3. Cytotoxicity test of 5-FU drug    31
Figure 4. Flow cytometry analysis of SW480 cells treated with different concentrations of DI peptide    32
Figure 5. Inhibition of apoptosis by pan-caspase inhibitor    33
Figure 6. Western blot analysis of apoptotic markers in SW480 cells    34
Figure 7. Western blot analysis of necrotic markers in SW480 cells    35
Figure 8. Western blot analysis of DNA fragmentation in SW480 cells    36
Figure 9. TUNEL assay on SW480 cells after treatment with different concentrations of DI peptide    37
Figure 10. Laser scanning confocal microscopy fluorescence images of DI peptide in different concentrations    39
6.    References    40
7.    Appendix    46
Appendix 1. HPLC analysis of DI peptide    46
Appendix 2. Mass spectroscopy analysis of DI peptide    47
Appendix 3. BCA standard curve    48
Appendix 4. Formula of solutions    49
                                

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