骨肉瘤是最常見的原發性惡性骨腫瘤且惡性程度最高，好發於兒童及青少年，而骨肉瘤的肺轉移通常是導致患者死亡的主因。chemokine (C-C motif) ligand 24 (CCL24) 為CC chemokine家族的一員，主要在發炎反應下作用，調控白血球的活動，與癌症的轉移跟侵襲以及血管新生的調控有關。褪黑激素 (Melatonin) 主要是腦部松果體 (Pineal gland) 所分泌的一種激素，具有調節生理時鐘的作用此外研究證實，褪黑激素具有抗氧化、抗發炎以及抗癌的功效，但褪黑激素對於骨肉瘤療效尚未被釐清。因此，本研究中主要探討褪黑激素是否會對骨肉瘤細胞的生長與轉移造成影響。首先以細胞毒性實驗觀察到褪黑激素在骨肉瘤細胞株（U2OS和HOS）中並無細胞毒性。接著以Wound healing Assay和boyden chamber Assay發現褪黑激素確實能抑制骨肉瘤細胞的爬行和侵襲能力。用gelatin zymography方式發現褪黑激素並不會影響MMP-9及MMP-2蛋白的活性。因此利用 RNA-Sequencing (RNA-seq) 找出褪黑激素可能影響轉移之相關基因CCL24，接著以Reverse transcription-polymerase chain reaction (RT-PCR)、real-time PCR、enzyme-linked immunosorbent assay (ELISA) 發現褪黑激素能有效抑制CCL24 mRNA及其分泌量。利用Western blot發現人類骨肉瘤細胞U2OS在處理褪黑激素後，是透過抑制磷酸化JNK1/2蛋白表現來抑制細胞的轉移。總結以上結果，我們發現在骨肉瘤細胞中，褪黑激素能抑制訊息傳遞路徑中的JNK1/2訊息傳遞路徑來降低CCL24的分泌，來達到抑制骨肉瘤細胞轉移和侵襲的能力。因此，褪黑激素可能可以用來預防骨肉瘤的轉移，並期望能應用在未來癌症的治療上。

Osteosarcoma is the most malignant and common primary bone tumor, occurring in developing children and adolescents. Lung metastasis of osteosarcoma is usually the leading cause of death in patients. Chemokine (C-C motif) ligand 24 (CCL24), a member of the CC chemokine family, a proinflammatory cytokine, usually act to induce the migration and activation of leukocytes in immune responses. Regulate cancer progression, especially the processes of invasion, metastasis and angiogenesis. Melatonin is a naturally occurring molecule mainly secreted by the pineal gland and known as a gatekeeper of circadian clocks. Many reports demonstrate that melatonin had an antioxidant, antiinflammatory and anti-cancer effect. However, the therapeutic potential of melatonin on osteosarcoma remains unclear. Therefore, we aimed to determine whether melatonin affects the growth and migration of osteosarcoma cells. In this study, we used osteosarcoma cell lines (U2OS and HOS) to conduct experiments and determine that the melatonin on cell viability. We also found the inhibitory of the migration and invasion ability of osteosarcoma cells by wound healing assay and boyden chamber assay. According to a gelatin zymography assay, increasing in the concentration of melatonin, didn’t affect protein levels of MMP-9 and MMP-2. Moreover, RNA sequencing (RNA-seq) found that CCL24 gene may affect metastasis. According to a reverse-transcription polymerase chain reaction (PCR), real-time PCR, ELISA to verify, revealed that melatonin inhibited CCL24 mRNA and CCL24 secretion. Furthermore, the effect of melatonin on the MAPK pathway and PI3K/AKT signallings were examined to show melatonin reduce the protein levels of JNK1/2, inhibits osteosarcoma cell metastasis. Based on these findings, we concluded that melatonin inhibited the activity of JNK1/2 protein levels and CCL24 secretion, lead to reduce the metastasis of osteosarcoma. In conclusion, melatonin may be a powerful candidate for a preventive agent against osteosarcoma invasion and metastasis.

摘要.................................................I
Abstract............................................II
縮寫表..............................................IV

第一章 緒論..........................................1
1. 骨癌.........................................1
2. 褪黑激素的介紹...............................6
3. 趨化激素的介紹...............................8
4. 腫瘤的轉移和侵襲............................10
5. 基質金屬蛋白水解酶..........................11
6. 訊息傳遞路徑................................12
7. 研究動機....................................14

第二章 實驗方法.....................................15
1. 實驗材料與配製..............................17
2. 細胞培養....................................24
3. 細胞存活率分析..............................26
4. 體外腫瘤轉移試驗............................26
5. 受質十二烷基硫酸鈉聚丙烯醯胺凝膠電泳........29
6. 西方點墨法..................................30
7. 反轉錄-聚合酶連鎖反應.......................31
8. RNA干擾實驗.................................33
9. 酵素連結免疫吸附法 (Enzyme linked-immunosorbent assay；ELISA) ......................................34
10. RNA定序 (RNA-sequencing) ...................35
11. 統計分析....................................35

第三章 結果.........................................36
1. 褪黑激素對於人類骨肉瘤細胞株 U2OS、HOS 之細胞存活率的影響..............................................36
2. 褪黑激素對於人類骨肉瘤細胞株 U2OS、HOS 之細胞移動能力影響..............................................36
3. 褪黑激素對於人類骨肉瘤細胞株 U2OS 及 HOS 爬行能力之影響................................................36
4. 褪黑激素對於人類骨肉瘤細胞株 U2OS 及 HOS 侵襲能力之影響................................................37
5. 褪黑激素對於人類骨肉瘤細胞株 U2OS 及 HOS 分泌 MMP-2 及 MMP-9 酵素活性之影響.............................37
6. 利用 RNA-Sequencing 來觀察褪黑激素對於人類骨肉瘤細胞株 U2OS mRNA表現量之影響............................37
7. 褪黑激素對於人類骨肉瘤細胞株 U2OS 其 CCL24 mRNA 表現量之影響............................................38
8. 褪黑激素對於人類骨肉瘤細胞株 U2OS 其 CCL24 於不同時間表現量之影響......................................38
9. 利用 CCL24 合成蛋白來觀察骨肉瘤細胞株 U2OS 及 HOS 轉移能力之影響........................................39
10. CCL24 對於人類骨肉瘤細胞株 U2OS 及 HOS 轉移及侵襲能力之影響............................................39
11. 褪黑激素對於人類骨肉瘤細胞株 U2OS 其 CCL24蛋白表現量之影響..............................................40
12. 褪黑激素對於人類骨肉瘤細胞株 U2OS 及 HOS 其訊息傳遞路徑之影響..........................................40


第四章 討論.........................................42

參考文獻............................................47
圖表與圖表說明......................................58

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