臺灣博碩士論文加值系統

黑色素瘤(melanoma)是起源於黑色素細胞的惡性腫瘤，屬於致死率極高且預後不良之皮膚癌，近年來，黑色素瘤的發病率在全球呈現增長的趨勢，開始成為一個引人矚目的醫療問題。表沒食子兒茶素沒食子酸酯[(-)-epigallocatechin-3-gallate, EGCG]為綠茶兒茶素中主要的生物活性成分，近年來已被許多的研究證實可有效抑制腫瘤的形成和發展，但侷限於高濃度EGCG對正常細胞的毒性反應及在人體內短暫的半衰期及高清除率，為使EGCG得以有效發揮更大的抗癌效果，本研究利用EGCG與物理方法製備而成之奈米金粒子(physical nanogold, pNG)，藉由兩者以特定比例混合且經超音波震盪，製成奈米等級之「表沒食子兒茶素沒食子酸酯奈米金粒子結合物」(以下簡稱為「EGCG-pNG」)，EGCG-pNG已被證實可表現出較單獨的EGCG更長的活性半衰期、更長的控制釋放時間及更佳的抗膀胱癌效果。本研究藉由小鼠B16-F10黑色素瘤細胞，進行EGCG-pNG之抗黑色素瘤效果評估。細胞實驗方面，相較於僅以EGCG加以處理之小鼠B16-F10黑色素瘤細胞，EGCG-pNG對小鼠B16-F10黑色素瘤細胞表現出高達4倍以上的細胞毒殺增強效果，而這樣的細胞毒殺增強效果主要是藉由粒線體途徑介導的細胞凋亡，並透過凋亡小體染色、JC-10染色及caspase-3, -8, -9之活性測定加以確定。在小鼠黑色素瘤動物模型實驗中，分別以EGCG-pNG與EGCG直接注入黑色素瘤模型小鼠之腫瘤部位時，在腫瘤大小之變化方面，EGCG-pNG相較於EGCG表現出1.66倍抑制腫瘤生長的效果。而在人類紅血球細胞之溶血試驗中，EGCG在與pNG進行結合後，有助減少pNG單獨與人類紅血球細胞作用下，血紅蛋白之釋放量，顯現其良好之生物相容性。綜上所述，證實EGCG-pNG具有增強EGCG對小鼠B16-F10黑色素瘤細胞的體外及體內之抗腫瘤效果。

Malignant melanoma, the major fatal form of skin cancer, is an aggressive and refractory cancer derived from melanocytes. In recent years, the incidence of melanoma has been continuously increasing worldwide and becoming a huge public health issue. (-)-Epigallocatechin- 3-gallate (EGCG), the major bioactive constituent in green tea, has been reported to effectively inhibit the formation and development of tumors. However, high concentrations of EGCG leading to the death of normal cells, short half-life and clearance rate of EGCG limit its application. To maximize the effectiveness of EGCG, EGCG and gold nanoparticles prepared from physical method (physical nanogold, pNG) were mixed at specific ratios and prepared via sonication to form nano-sized conjugate of EGCG and pNG (EGCG-pNG). EGCG-pNG has been proved to be longer activity half-life, longer controlled release time and better anti-cancer effect on bladder cells than EGCG in vitro and in vivo. In this study, evaluation of anti-melanoma efficacy via EGCG-pNG was evidenced from murine B16-F10 melanoma cells. As a result, EGCG-pNG showed the better anticancer efficacy than EGCG in B16F10 murine melanoma cells, in which the cytotoxic effect in the melanoma cells treated with EGCG-pNG was over four times higher than those treated with EGCG only. The enhance¬ment is achieved through mitochondrial pathway- mediated apoptosis as determined by staining of apoptotic bodies, JC-10 staining, and caspase-3, -8, -9 activity assay. Moreover, EGCG-pNG was 1.66 times more potent than EGCG for inhibition of tumor growth in a murine melanoma model. In the hemolysis assay, the pNG surface conjugated with EGCG is most likely the key factor that contributes to the decreased release of hemoglobin from human red blood cells. Overall, these findings implicated that EGCG-pNG could enhance the anti-cancer efficacy of EGCG for murine B16-F10 melanoma cells in vitro and in vivo.

目錄 I
表目錄 III
圖目錄 IV
附錄目錄 VI
摘要 VII
Abstract IX
第壹章、緒言 1
第貳章、文獻整理 5
一、黑色素瘤(melanoma) 5
I-1.危險因子 6
I-2.不同時期之存活率 10
I-3.預防 14
I-4.治療 15
I-5.小結 22
二、表沒食子兒茶素沒食子酸酯[(-)-epigallocatechin-3-gallate,EGCG]與癌症 24
II-1.抗癌機轉 26
II-2.EGCG與67kDa層粘連蛋白受體(laminin receptor)的作用 30
三、奈米粒子藥物 32
III-1.奈米金粒子 34
III-2.靶向給藥系統與癌症治療 40
III-3.EGCG-pNG之靶向給藥策略 49
第參章、實驗設計 53
一、細胞試驗(in vitro) 53
二、動物試驗(in vivo) 54
第肆章、材料與方法 55
一、實驗材料 55
I-1.細胞株 55
I-2.小鼠 55
I-3.表沒食子兒茶素沒食子酸酯[(-)-epigallocatechin-3-gallate, EGCG] 55
I-4.物理性奈米金 56
I-5.Taxol(太平洋紫杉醇) 56
I-6.Z-VAD-FMK 56
I-7.細胞培養及凍存相關試劑 56
I-8.組織及細胞染劑 57
I-9.試劑組 57
I-10.儀器設備 57
二、實驗方法 59
II-1.兒茶素-奈米金粒子(EGCG-pNG)製備 59
II-2.兒茶素-奈米金粒子(EGCG-pNG)基本性質分析 59
II-3.細胞培養(cell culture) 60
II-4.細胞毒殺實驗(cytotoxicity assay) 60
II-5.細胞粒線體膜電位變化檢測實驗 (JC-10 staining assay) 62
II-6.Annexin V/ PI 分析實驗 63
II-7.caspase 3、8、9之活性試驗 65
II-8.活體B16-F10黑色素瘤生長抑制實驗 66
II-9.人類紅血球溶血試驗 67
第五章、結果與討論 71
一、EGCG-pNG之結合原理 71
二、EGCG-pNG特性分析 73
三、EGCG-pNG可增強EGCG對小鼠黑色素瘤細胞的體外抗腫瘤作用 74
四、EGCG-pNG可藉細胞凋亡路徑增強EGCG對小鼠黑色素瘤細胞的體外抗腫瘤作用 82
五、EGCG-pNG可增強EGCG對小鼠活體內之抗黑色素瘤效果 88
六、EGCG-pNG與人類紅血球細胞間具良好之生物相容性 90
七、EGCG-pNG之抗癌潛力 92
第陸章、結論 95
第柒章、參考文獻 97

表目錄
Table 1.Four major catechin formulas and molecular weights 123
Table 2.Synthetic methods and for gold nanoparticles of different core sizes 124
Table 3.Anticancer substances conjugated with gold nanoparticles 125
Table 4.Escape strategies for biological barriers in nanoparticle-based cancer therapies 126
Table 5.B16-F10 and Vero cell viabilities after 24 hours treatment 127
Table 6.IC50 of pNG and EGCG on Vero and B16-F10 cell proliferation after 24 hours treatment 128
Table 7.24 hours average B16-F10 cells cytotoxicity via EGCG-pNG 129
Table 8.Characterization of EGCG-pNG versus cell viabilities after 24 hours treatment 130
Table 9.Average tumor volumes of C57/BL6 male mice after intratumoral treatment with PBS, EGCG and EGCG-pNG 131
Table 10.3 hours hemolysis level of EGCG-pNG, pNG and EGCG 132


圖目錄
Fig. 1 Structure of the major catechins found in green tea. 133
Fig. 2 Applications of gold nanoparticles in Biomedical field. 134
Fig. 3 The effects of EGCG on Vero and B16-F10 cell proliferation. 135
Fig. 4 The effects of pNG on Vero and B16-F10 cell proliferation. 136
Fig. 5 Direct observation of improving anti-proliferative activity of EGCG and pNG in vitro. 137
Fig. 6 B16-F10 cell viabilities after 24 hours treatment. 138
Fig. 7 Comparision of cytotoxicity effects between the mixtures of EGCG and pNG with or without ultrasound pretreatment. 139
Fig. 8. Evaluation of enhanced apoptosis induction of EGCG by pNG via JC-10 staining in B16-F10 cells. 140
Fig. 9 Evaluation of enhanced apoptosis induction of EGCG by pNG via Annexin V &; PI staining in B16-F10 cells. 141
Fig. 10 Evaluation of enhanced apoptosis induction of EGCG by pNG via caspase-3, -8, and -9 activities in B16-F10 cells. 142
Fig. 11 Initiation of time-dependent apoptotic activation by EGCG-pNG treatment via caspase pathway in B16-F10 melanoma cells. 143
Fig. 12 Melanoma mice model protocol and direct observarion of increased tumor growth inhibition of EGCG by pNG in vivo. 144
Fig. 13 Increased tumor growth inhibition of EGCG by pNG via histology evaluation in vivo. 145
Fig. 14 Increased tumor growth inhibition of EGCG by pNG via tumor volumes evaluation in vivo. 146
Fig. 15 Photographs of the RBCs hemolysis after treatment of EGCG. 147
Fig. 16 Photographs of the RBCs hemolysis after treatment of pNG. 148
Fig. 17 Photographs of the RBCs hemolysis after treatment of EGCG-pNG. 149
Fig. 18 Hemolysis results reported as a percentage of the positive control condition. 150


附錄目錄
Appendix 1. Relationship of the atoms percentage at the surface and particle size in the gold nanoparticles 151
Appendix 2. Publication of dissertation 152

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