臺灣博碩士論文加值系統

Survivin為IAP家族成員之一，其具有調控細胞分裂以及抑制細胞凋亡的功能。在正常細胞中Survivin主要大量表現在細胞分裂的G2/M時期，但是survivin在許多不同種類的癌細胞中都有大量表現的現象。在腫瘤細胞中survivin的大量表現會影響病人對化療藥物的抗藥性並有較差的預後。除此之外，近年來的研究也陸續發現survivin具有調控細胞移行和癌症轉移的功能，然而survivin對於細胞移行的相關機制目前仍不明確。在本研究中我們利用shRNA抑制原本survivin表現量較高且移行能力較快的食道癌細胞株TE-9的survivin表現量，結果觀察到細胞的移行能力明顯的受到抑制。同時，我們在爬行能力較弱的CE48T/VGH和TE-2食道癌細胞株中大量表現HA-survivin，也觀察到細胞的移行能力有上升的現象，證明survivin具有正向調控細胞移行的能力。進一步分析survivin對細胞侵襲能力的影響，發現在TE-9細胞株中抑制survivin的表現量也降低了細胞的侵襲能力，並可觀察到金屬基質蛋白酶MMP-2的表現量上升。先前研究指出survivin可能透過NF-κB的訊息傳遞路徑來影響到細胞的移行和侵襲能力，利用西方墨點法分析NF-κB 上游調控分子IκB-α的表現量，發現在TE-9 細胞中抑制survivin的表現會導致IκB-α的表現量上升，進而影響NF-κB在細胞核中的分佈比例，此外，在TE-9細胞株中抑制survivin可以觀察到Akt的蛋白質表現量減少進而影響Akt的訊息傳遞，而Akt的訊息傳遞同時也會調控NF-κB，因此我們推測survivin可能透過Akt/ NF-κB 的訊息傳遞路徑來調控MMP-2的表現量進而影響細胞侵襲的能力。Aurora-A是一個絲胺酸/蘇胺酸激酶，Aurora-A過去也被報導和細胞分裂、抑制細胞凋亡、促進細胞爬行和侵襲有關，在我們實驗室先前研究也發現Aurora-A能夠磷酸化survivin並增加survivin的蛋白質穩定度以調控survivin抑制細胞凋亡的功能。但是Aurora-A是否調控survivin對細胞移行及侵襲能力的影響目前仍未知。在TE-2細胞株中大量表現Aurora-A雖然不影響細胞的侵襲能力，但仍可能調控細胞的移行能力，後續實驗將繼續探討Aurora-A對細胞移行能力的影響，並進一步探討Aurora-A對survivin的磷酸化在survivin調控細胞移行的功能上可能扮演的角色。

Survivin, a member of inhibitor of apoptosis protein (IAP) family, has dual functions in cell cycle regulation and anti-apoptosis. Survivin is markedly expressed in the G2-M phase of cell cycle in normal cells but highly expressed in various human cancers. The presence of survivin in tumors is associated with chemoresistance and poor prognosis. Moreover, survivin has been reported to promote tumor cells migration and cancer metastasis in recently years. However, the role of survivin in esophageal cancer cell migration is still unclear. In this study, we used transwell migration assay to determinate the effect of survivin on cell migration. Knockdown survivin by shRNA in TE-9 cells, which has highly endogenous survivin expression, decreased cell migration significantly. Conversely, overexpression of survivin in CE48T/VGH and TE-2 cells promoted cell migration. Furthermore, we found that knockdown survivin in TE-9 cells decreased cell invasion by reduced MMP-2 expression. Previous study have shown the strong correlation between survivin and NF-κB signaling pathway, we also shown that knockdown surviving in TE-9 cells elevated IκB-α then inhibited NF-κB nuclear translocation. In addition, knockdown survivin in TE-9 cells decreased the protein level of Akt, which may activated NF-κB pathway. These suggest that knockdown survivin downregulates MMP-2 expression through Akt/ NF-κB pathway inhibition. In our lab, we haves found that survivin was phosphorylated at Thr21 and Ser81 by a mitotic serine/threonine kinase Aurora-A, which plays an important role in cell division, anti-apoptosis and cell migration. This phosphorylation increased survivin protein stability but also sustained the function of survivin to inhibit apoptosis. However, overexpression Aurora-A in TE-2 cells did not regulate cell invasion. We will further investigate the effect of Aurora-A on cell migration regulation. Additionally, whether Aurora-A mediated phosphorylation is required for the function of survivin to promote migration also will be studied.

Abstract……i
中文摘要……ii
目錄……iii
緒論……1
一、食道癌介紹……1
二、Survivin……2
三、Aurora-A……4
四、Survivin與Aurora-A的關係……5
五、研究目的……7
實驗材料……8
一、細胞培養……8
二、引子設計……8
三、抽取質體……9
四、質體……9
五、慢病毒……9
六、腺病毒……9
七、細胞移行能力與侵襲能力測定……10
八、蛋白質萃取與定量……10
九、西方墨點法……10
十、免疫螢光染色……12
實驗方法……13
一、細胞培養……13
二、建構帶有HA-survivin之慢病毒質體……13
三、抽取質體……14
四、腺病毒製備……15
五、腺病毒感染……16
六、慢病毒感染……17
七、細胞移行能力測定……17
八、細胞侵襲能力測定……18
九、蛋白質萃取與定量……18
十、SDS-聚丙烯醯胺膠體電泳 (SDS-PAGE)……19
十一、免疫螢光染色……19
實驗結果……21
一、Survivin對食道癌細胞移行能力的影響……21
二、Survivin對食道癌細胞侵襲能力的影響……21
三、Survivin影響細胞侵襲能力的訊息傳遞路徑……22
四、大量表現Aurora-A不影響食道癌細胞侵襲能力……23
討論……24
參考文獻……29
圖……34

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