Project 1中文摘要
根據我國2013年國人兩性十大癌症死因死亡率的統計資料中可以看到子宮頸及部位未明示子宮癌相關的癌症在女性的統計資料中位居第七位，說明其對女性健康的威脅，是值得我們關注的議題。
而目前治療子宮肉瘤癌(uterine sarcoma)的方式有，手術移除有發生癌病變的區域；以及對患部進行放射線治療；和對病人投與化療藥物進行治療；或是進行賀爾蒙治療。而近年來癌症治療中已被受討論的是化療藥物所產生的抗藥性問題。而在治療子宮肉瘤癌中，最常使用的藥物為艾黴素(doxorubicin)，雖然已有很多報導指出部分與抗藥性相關的生物機制，但其不能完全的說明產生抗藥性的原因，因此對艾黴素有抗藥性的子宮肉瘤癌的機制仍是值得被探討的。
由實驗室以前的研究知道在具抗藥性的子宮肉瘤癌細胞株(MES-SA/DxR-8μM)中發現RCN1和XRCC3這兩個蛋白的表現量與不具抗藥性的子宮肉瘤癌細胞株(MES-SA)相比其蛋白表現量分別相差了2.06倍以及2.13倍。有趣的是，實驗進行中在具抗藥性的子宮肉瘤癌細胞株的序列發現了RCN1_T5G與XRCC3_T241C這兩個點突變(point mutation)，於是我將這兩個點突變進行reverse point mutation，並將實驗所得到的四個表現質體(RCN1, RCN1_T5G, XRCC3, XRC3_T241C)分別轉染於不具抗藥性的子宮肉瘤癌細胞來探討其對癌細胞抗藥性的影響。實驗結果發現，過度表現XRCC3_T241C質體的細胞其對艾黴素的抗藥性有顯著的增加，且會降低細胞的凋亡比例；這說明了XRCC3 Thr241Met的突變與抗藥性的子宮肉瘤癌有顯著的相關。而RCN1的部分因為質體轉染入不具抗藥性的子宮肉瘤癌細胞後，其蛋白的表現量並未增加，因此會持續的進行細胞篩選，以篩選出有表現差異的細胞。希望這個研究的結果未來能夠提供給子宮肉瘤癌抗藥性相關的研究一個有用的參考。

Project 2中文摘要
根據衛生福利部統計的國人十大死因中惡性腫瘤32年來一直蟬連榜首，其中口腔癌的死亡率是位居台灣2013年的十大癌症死因死亡率統計資料第五位，而且口腔癌好發於男性，男女性別比大約介於3：1至2：1[4]。和所有的癌症一樣，口腔癌的高死亡率和口腔癌細胞的侵襲、轉移能力息息相關。可惜的是，統計資料指出，超過六成以上的口腔癌患者在就醫時，其癌細胞就已經發生區域性或遠端的轉移，而其病例的五年存活率與未發生轉移的口腔癌患者相比，明顯地大幅下降了50%[32]。因此癌症的可怕之處除了在於癌細胞會不受控制的增生外，更危險的是癌細胞所產生的轉移問題。由過去的文獻指出在成纖維細胞以及骨肉瘤癌細胞中，其基因表現分析中發現SCaMC-1蛋白有過度表現[33]。而本研究希望利用核糖核酸干擾(RNA interference, RNAi)的技術藉由siSCaMC-1後以體外Transwell侵襲能力測試(Transwell invasion ability assay)觀察細胞的侵襲能力，以體外傷口癒合實驗(Wound healing migration assay)來觀察細胞的移動能力，以及細胞存活率測試(MTT cell viability assay)、細胞週期測試(Cell Cycle assay)來觀測細胞的存活能力，來觀察干擾SCaMC-1蛋白表現是否會使得具轉移能力的口腔癌細胞OC3-I5其細胞侵襲、轉移、存活能力下降，以限制癌細胞轉移能力並達到降低惡性腫瘤的死亡率。本研究也進一步利用西方點墨法(Western blot)探討SCaMC-1蛋白對上皮細胞間質轉化(Epithelial-mesenchymal transition, EMT)過程的影響。由實驗結果發現siSCaMC-1後，上皮細胞間質轉化因子Snail、Twist、SIP1蛋白表現量減少，進而升高E-cadherin蛋白的表現量，減少Vimentin、MMP3，以及MMP7蛋白的表現量，導致細胞的侵襲移動能力下降、減少走向上皮細胞間質轉化，進一步也會降低細胞的存活率。因此我們推論在口腔癌細胞中過度表現的SCaMC-1蛋白很有可能有助於癌細胞走向上皮細胞間質轉化。希望藉此研究了解SCaMC-1發展為口腔癌的診斷和治療新標靶蛋白的潛力。

Project 1 Abstract
According to Taiwan's Ministry of Health and Welfare Statistics of top ten cancer causes of death in 2013, the cervix and uterine related cancers is the seventh leading cause for females. Therefore, the risk of the cervix and uterine related cancers to women’s health is an issue worthy of our attention. Current treatments of uterine sarcoma manner are surgery, radiation therapy, chemotherapy, and hormone therapy. In recent studies of chemotherapy, the causes and burden of drug resistance have been in-depth discussed. In the treatment of uterine sarcoma, the most commonly used drug is doxorubicin. Although the biological mechanisms associated with doxorubicin resistance has been widely reported, the precise mechanism of resistance remains unclear. Therefore, doxorubicin resistance forming mechanisms still deserves to be explored. Previous studies have known that RCN1 and XRCC3 protein expression in MES-SA/DxR-8μM cells are higher 2.06 times and 2.13 times than MES-SA cells, respectively. Unexpectedly, RCN1_T5G and XRCC3_T241C point mutations were found in MES-SA/DxR-8μM cells that we decide to reverse the mutation sites back. Therefore, the purpose of the study is to transfect four different overexpression plasmids (RCN1, RCN1_T5G, XRCC3, XRCC3_T241C) in MES-SA cells to investigate the role of mutation sites in drug resistance by means of cells proliferation and cell apoptosis. My experimental results showed that resistant ability of MES-SAXRCC3_T241C cell to doxorubicin has significantly increased, and cell apoptosis mediated has decreased. This result explains that XRCC3 Thr241Met polymorphism significant association with doxorubicin-resistance in uterine sarcoma. However, the RCN1_T5G and RCN1 plasmids are still underway to be transfected into MES-SA cells. I will continue to optimize G418 concentration to select cells that can stably express transfected DNA. I wish that this study can provide a useful reference to doxorubicin resistance in future.

Project 2 Abstract
According to Taiwan's Ministry of Health and Welfare Statistics in 2013, oral cancer is the fifth leading cause of death in cancer. The incidence rates of oral cancer in males are 2~3 times higher than in females. Since approximately nine in ten of cancer deaths are attributable to cancer metastasis, cancer metastasis is a main cause of failure treatment. When patients present with oral cancer, over 60% of them have spreaded cancer cells in regional lymph nodes or distant organs at their first medical care. The five-year survival rates of these patients compared with cancer cells confined to primary site (localized) decrease as high as 50%. Recently, the comprehensive gene expression analysis showed that SCaMC-1 overexpression is a general feature of transformed cancer cells. In this study, we used a pair of oral squamous cell carcinoma lines, OC3, and invasive OC3-I5 as a model system to examine invasive mechanism and to identify whether SCaMC-1 protein is a therapeutic target. We used RNA interference technique to monitor the influence of SCaMC-1 protein in cell proliferation, migration and invasion ability. Our results show that siSCaMC-1 can reduce invasive OC3-I5 cells cell proliferation, migration and invasion. We also used western blotting to examine SCaMC-1 protein’s role in Epithelial-mesenchymal transition (EMT) pathway. The experimental results showed that after siSCaMC-1, reducing epithelial-mesenchymal transition factor Snail, Twist, SIP1, and further, increasing E-cadherin, reducing Vimentin, MMP3, MMP7 protein expression, leading to decreased cell motility, EMT and cell survival. Therefore, we infer in oral cancer cells, SCaMC-1 overexpression may likely to contribute to epithelial-mesenchymal transition (EMT). Taken together, our preliminary results demonstrate that SCaMC-1 protein is a useful diagnostic marker and therapeutic target for reducing oral cancer metastasis.

目錄 i
表目錄 iv
圖目錄 v
誌謝 vii
縮寫清單 ix
Project 1：Roles of RCN1 and XRCC3 in Doxorubicin-Resistant Uterine Cancer 1
中文摘要 2
英文摘要 3
壹、文獻探討 4
一、子宮肉瘤癌(Uterine sarcoma) 4
二、艾黴素(Doxorubicin) 9
三、Reticulocalbin-1 (RCN 1) 11
四、XRCC3 (X-ray repair complementing defective repair in Chinese hamster cells 3) 13
五、突變(Mutation) 15
六、研究目的 17
貳、材料與方法 18
一、藥品試劑與材料 18
二、細胞樣品製備 20
三、過度表現和轉染的製程(Overexpression stable clone and transfection) 21
四、Recover point-mutation的製程 22
五、利用慢病毒過度表現和感染的製程(Overexpression stable clone, lentiviral vector preparation and infection) 23
六、蛋白質表現差異的驗證 24
七、細胞存活率測試(MTT cell viability assay) 26
八、細胞凋亡(Cell apoptosis) 27
參、實驗結果 28
一、引子(Primer)的設計 29
二、利用PCR來測試最佳作用黏合(annealing)溫度 31
三、PCR產量放大、產物凝膠提取(Gel extraction) 32
四、利用限制酶進行DNA digestion切位確認、產物Clean up 33
五、進行pCMV6 vector與RCN1、XRCC3 insert接合(Ligation) 34
六、RCN1、XRCC3轉形(Transformation) 35
七、進行菌盤單一菌落之挑菌、搖菌與抽取小量質體進行確認 36
八、將質體送定序的結果與NCBI中的序列進行比對 38
九、Reverse point mutation引子(Primer)的設計 40
十、進行Reverse point mutation的PCR 42
十一、進行Reverse point mutation PCR產物的Clean up以及DpnI digestion 43
十二、Reverse RCN1_T5G、Reverse XRCC3_T241C進行轉形(Transformation) 44
十三、菌盤單一菌落之挑菌、搖菌與抽取小量質體進行確認 45
十四、將Reverse完成的質體定序結果與NCBI中的序列進行比對 46
十五、XRCC3_T241C以及XRCC3質體進行載體置換 48
十六、利用西方點墨法確認Lipofectamine過度表現RCN1的效率 49
十七、利用西方點墨法確認Lenti-virus過度表現XRCC3的效率 50
十八、比較轉染入不同質體間的子宮肉瘤癌細胞之生長速率 51
十九、利用艾黴素測試轉染入不同質體的細胞其毒殺的效果 52
二十、利用Flow cytometry測試轉染入不同質體的細胞其凋亡現象 54
二十一、利用Western blot觀察細胞與凋亡蛋白之間的調控關係 55
肆、討論 56
伍、結論 58
Project 2：Role of SCaMC-1 in metastatic oral cancer cell line 59
中文摘要 60
英文摘要 61
壹、文獻探討 62
一、口腔癌 62
二、癌症轉移 66
三、RNA干擾(RNA interference, RNAi)技術 69
四、研究目的 71
貳、材料與方法 72
一、藥品試劑與材料 72
二、樣品製備 74
三、蛋白質表現差異的驗證 76
四、利用siRNA knockdown目標蛋白 78
五、體外Transwell侵襲能力測試 (Transwell invasion ability assay) 79
六、體外Transwell遷移能力測試 (Transwell migration ability assay) 80
七、傷口癒合實驗(Wound healing migration assay) 81
八、細胞存活率測試(MTT cell viability assay) 82
九、細胞週期測試(Cell Cycle assay) 83
參、實驗結果 84
一、比較OC3和OC3-I5細胞的侵襲、轉移能力 84
二、SCaMC-1在口腔癌細胞中knockdown效率確認 88
三、觀察siSCaMC-1對於口腔癌細胞侵襲能力的影響 92
四、觀察siSCaMC-1對於口腔癌細胞移動能力的影響 93
五、觀察siSCaMC-1對於口腔癌細胞存活能力的影響 95
六、利用流式細胞儀分析siSCaMC-1對於口腔癌細胞的影響 96
七、利用西方點墨法分析siSCaMC-1對於EMT pathway的影響 97
肆、討論 98
伍、結論 100
參考資料 103
附 錄 108
壹、Hyperglycemia initiates N-cadherin rearrangement and diabetic monocytes promote inflammatory responses in human microvascular endothelial cells短文簡介 109

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