臺灣博碩士論文加值系統

癌症居國內十大死因之首位，其中腎臟癌是泌尿腫瘤中導致死亡率的第三名，平均約有百分之30的病人因癌細胞之轉移而致命，故若能有效抑制癌細胞的生長或轉移，將可大幅減低癌症的死亡率。因此發展新穎天然藥物來當作癌症預防或是治療藥物是目前最重要的課題。Nimbolide是第一個從印度楝 (Azadirachta indica)的葉和花提煉的天然物，目前Nimbolide被發現的生物功能中包括：抗轉移、抗癌、抗發炎、抗氧化和細胞凋亡等多重功用。但是，目前Nimbolide對於人類腎臟癌細胞的作用機制，至今仍然未知。
我們實驗結果以MTT和Annexin/V染色方式測試不同濃度Nimbolide處理人類腎臟癌細胞(786-O)作用24小時，證實Nimbolide會抑制786-O細胞的生長、誘導細胞週期停留G2/M期、造成DNA損傷和誘導細胞凋亡，同時也會造成cyclin A、cyclin B、cdc2、cdc25c蛋白下降，p-cdc2、p-cdc25c和p53表現上升。另外，也會造成caspases-3、-9、PARP、DR5、CHOP 和 Bax活化，抑制Bcl-2和Mcl-1蛋白下降，因此我們認為Nimbolide會誘導細胞凋亡。腫癌細胞侵襲作用通常伴隨一些生理變化，其中包含細胞基質瓦解和調控細胞移動作用。我們採用細胞移動和侵襲方式證實Nimbolide抑制786-O細胞移動和侵襲能力，以gelatin/casein zymography、西方墨點法方式證實Nimbolide會抑制uPA蛋白表現和活性，但不影響MMP-2和MMP-9表現。希望未來Nimbolide在腎臟癌方面當作抗癌藥物和抗侵襲藥物的發展上深具潛力。

Cancer is the top of ten causes of death in Taiwan, especially renal cell carcinoma (RCC) is the third leading cause of death among urological tumours. The metastasisof cancer cells leads 30% patients died, and inhibition of cancer cells metastasis will markly decrease the death rate. Therefore, development of novel nature drug or drug therapy was important topic. Nimbolide was first derived from the leaves and flowers of neem (Azadirachta indica), a tree in the mahogany family. Nimbolide is a a wide range of pharmacological properties including anti-metastatic, anti-inflammatory, apoptosis and anti-oxidant effects. However, Nimbolide effect molecular mechanism of human renal cancer cells is presently unknown.
In this study, MTT assay suggested that Nimbolide inhibit 786-O cell growth, cell cycle arrest at G2/M phase, DNA damage and apoptosis by Comet assay, Annexin V/PI stain and flow cytometry analysis. Nimbolide inhibit the cyclin A、cyclin B、cdc2 and cdc25c expression, increases of p-cdc2, p-cdc25c and p53 expression. In addition, Nimbolide triggered the activations of caspases-3, -9, PARP, DR5, CHOP and Bax expression, downregulation of Bcl-2 and Mcl-1 expression, resulting in apoptosis induction. Tumor metasatasis are multifaceted processes that mainly involve proteolytic degradation of the extracellular matrix and regulate cell motility. In our study suggested that Nimbolide inhibit cell migration and invasion by cell migration and invasion assay. Gelatin/casein zymography and western blotting suggest that Nimbolide suppress the protein activity expression of uPA, but not effect of MMP-2 and MMP-9 expression. This study demonstrated that Nimbolide may have potential as anti-cancer and anti-invasion agents in human renal cancer cells.

壹、 中文摘要‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 4
貳、 英文摘要‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 5
參、 前言‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 6
一. 腎臟癌簡介…………………………………………… 6
二. 細胞週期……………………………………………… 9
三. 細胞移動、侵襲………………………………………13
四. MMP family…………………………………………….15
五. MAPK pathway………………………………………...17
六. Nimbolide介紹………………………………………...19
肆、 動機‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧23
伍、 實驗方法‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧24
一. 人類腎臟癌細胞繼代培養……………………………24
二. 細胞生長速率測定……………………………………24
三. 細胞週期分析…………………………………………25
四. 西方墨點法……………………………………………26
五. Comet assay…………………………………………….27
六. Annexin V/PI染色實驗………………………………..28
七. 細胞爬行/侵襲實驗…………………………………...28
八. MMPs活性分析………………………………………..29
九. uPA活性分析…………………………………………..30
十. 統計分析………………………………………………30
陸、 實驗結果‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧31
一. Nimbolide對於人類腎臟癌和正常腎小管細胞存活率的影響…………………………………………...……………….31
二. Nimbolide是否影響人類腎臟癌(786-O)的細胞週期的變化…………………………………………...……………….31
三. Nimbolide誘導人類腎臟癌(786-O)細胞週期G2/M相關蛋白表現………………………………………...…………….32
四. Nimbolide誘發人類腎臟癌(786-O)細胞DNA損傷的現象…...……………………………………………………….32
五. Nimbolide誘發人類腎臟癌(786-O)細胞凋亡的現象…..32
六. Nimbolide誘導人類腎臟癌(786-O)細胞凋亡蛋白表現….33
七. Nimbolide對於人類腎臟癌(786-O)細胞內源性和外源性蛋白影響……………………………………………………34
八. Nimbolide調控人類腎臟癌(786-O)細胞移動和侵襲作用.34
九. Nimbolide對786-O細胞分泌MMP-9、MMP-2和uPA表現量的影響……………………………………………………35
柒、 討論‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧36
捌、 實驗圖表‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧40
玖、 參考文獻‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧49

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