子宮頸癌是台灣女性最常見的癌症之一，並且放射治療在其扮演重要之角色。由文獻得知，影響放射線敏感度的因素與DNA傷害修復及細胞週期有關。我們的研究使用子宮頸癌細胞株測試指出無論半乳醣凝集素一(Galectin-1)存在與否，放射敏感性皆會受到H-Ras訊息傳遞路徑影響。輻射照射經降低Galectin-1表現量之HeLa細胞會降低其腫瘤克隆生成。若輻射照射經增加galectin-1表現量之HeLa及C33A細胞皆增加其腫瘤克隆生成。然而，輻射照射經降低H-Ras表現量之HeLa及C33A細胞皆不影響其腫瘤克隆生成。降低HeLa細胞Galectin-1表現量會抑制其因輻射所誘發的Raf-1及ERK之磷酸化反應，增加C33A細胞Galectin-1表現量會增強其因輻射所誘發的Raf-1及ERK之磷酸化反應。使用彗星檢測試劑以及偵測γ-H2AX表現量顯示，Galectin-1會降低此兩株細胞因輻射照射所誘發的DNA損傷。ROGDI是一個經調控DNA損傷指標(γ-H2AX)活性進而與基因體穩定性有關之分子，一般來說，G2/M DNA損傷的檢查關鍵時期對輻射較為敏感，然而，G1/S過渡階段對輻射較具抵抗性。在HeLa和C33A中，降低ROGDI表現量將抑制CDK 1/2、cyclin A/B表現量進而造成細胞停滯於G2/M過渡時期。除此之外，使用流體細胞儀及腫瘤克隆生成技術偵測此兩株細胞輻射照射後數據顯示，降低此兩株細胞ROGDI表現量會使細胞停滯於G2期且降低其生存率。經由以上發現顯示，Galectin-1需依賴性通過H-Ras引發之DNA修補訊息傳遞路徑進而調控放射耐受性，且降低ROGDI表現量驅使細胞週期停滯於最具放射敏感性的階段(G2/M)以及藉由γ-H2AX活性指數增加顯示此階段DNA亦承受嚴重性的損傷。

Cervical cancer is common cancer among women in Taiwan and Radiotherapy plays an important role. The literature indicates that radio-sensitivity is correlated to DNA repair and cell cycle. Our study tests whether Galectin-1 is involved in the radiosensitivity mediated by the H-Ras signaling pathway using cervical carcinoma cell lines. The knockdown of Galectin-1 expression in HeLa cells decreased clonogenic survival following irradiation. The clonogenic survival increased in both HeLa and C33A cells with Galectin-1 overexpression. The overexpression or knockdown of Galectin-1 did not alter radiosensitivity, whereas H-Ras was silenced in both cell lines. The knockdown of Galectin-1 inhibited the radiation-induced phosphorylation of Raf-1 and ERK in HeLa cells. Overexpression of Galectin-1 enhanced the phosphorylation of Raf-1 and ERK in C33A cells following irradiation. Galectin-1 decreased the DNA damage detected using comet assay and γ-H2AX in both cells following irradiation. ROGDI is associated with genome stability by regulating the activity of a DNA damage marker, γ-H2AX. In general, the G2/M DNA damage checkpoint is more sensitive to radiation, whereas the G1/S phase transition is more resistant to radiation. The downregulation of ROGDI led to a decreased expression of CDK 1/2 and cyclin A/B and resulted in a G2/M phase transition block in both HeLa and C33A cells. In addition, the downregulation of ROGDI increased cell accumulation at the G2 phase as detected using flow cytometry and decreased cell survival as revealed by clonogenic assay in HeLa and C33A cells following irradiation. These findings suggest that Galectin-1 mediates radioresistance through the H-Ras-dependent pathway involved in DNA damage repair and the downregulation of ROGDI can mediate radiosensitivity by blocking cells at G2/M, the most radiosensitive phase of the cell cycle, as well as exerting deleterious effects in the form of DNA damage, as shown by increased γ-H2AX activation.

論文審定書………………………………………………………………i
致謝………………………………………………………………………ii
中文摘要…………………………………………………………………iii
英文摘要…………………………………………………………………iv
第一章 前言………………………………………………………………1
1.1 醣蛋白分子與腫瘤細胞放射敏感性的相關性……………………..1
1.2 半乳醣凝集素一 (Galectin-1)………………………………….......2
1.3 ROGDI, 新的未知功能分子ROGDI………………………………..3
1.4 細胞週期與癌細胞放射線敏感度的關係…………………………..4
1.5 輻射離子傷害誘發γ-H2AX表現的意義…………………………....5
1.6 研究目的與未來展望………………………………………………..5
第二章 材料與方法………………………………………………………7
2.1 材料…………………………………………………………………..7
2.1.1 藥物與試劑……………………………………………………......7
2.1.2 抗體………………………………………………………….........8
2.1.3 細胞株……………………………………………………….........8
2.1.4 小片段干擾核糖核酸(siRNA)……………………………….......8
2.1.5 小片段髮夾型干擾核糖核酸(shRNA)………………………......9
2.1.6 質體…………………………………………………………........9
2.1.7 西方點墨法分析材料………………………………………........9
2.1.8 實驗套組……………………………………………………........9
2.2 方法………………………………………………………………...10
2.2.1 細胞培養…………………………………………………….......10
2.2.2 轉染siRNA進行基因剔除實驗……………………………........10
2.2.3 轉染shRNA進行基因剔除實驗……………………………......11
2.2.4 轉染表現質體……………………………………………….......12
2.2.5 應用流式細胞偵測儀偵測綠色螢光蛋白表現…………….......12
2.2.6 轉染持續活化H-Ras基因於HeLa細胞…………………….......13
2.2.7 免疫沉澱及西方點墨法…………………………………….......14
2.2.8 H-Ras活性測試……………………………………………….....15
2.2.9 放射處理及克隆測試……………………………………….......15
2.2.10 彗星偵測………………………………………………….........15
2.2.11 細胞增生及克隆生成測試……………………………….........16
2.2.12 應用流式細胞儀偵測細胞週期………………………….........16
2.2.13 應用原位鄰邊連接測試方法(PLA)檢測蛋白間
交互作用………………………………………………….........17
2.2.14 應用原位鄰邊連接測試方法(PLA)檢測單一蛋
白質表現量……………………………………………….........18
2.2.15 應用共軛焦顯微鏡觀察Galectin-1-GFP融合蛋白
分佈……………………………………………………….........18
2.2.16 質體製備………………………………………………….........19
2.2.17 RNA萃取及聚合酶連鎖反應分析……………………….........19
2.2.18 統計方法………………………………………………….........20
第三章 結果……………………………………………………………..21
3.1 調控子宮頸癌細胞中Galectin-1表現量…………………………..21
3.2 Galectin-1與HeLa及C33A的抗輻射敏感性有關…………………21
3.3 H-Ras在子宮頸癌抗輻射敏感性扮演顯著的角色，而非
K-Ras………………………………………………………………..22
3.4 Galectin-1藉由增強H-Ras訊息傳遞達到子宮頸癌細胞抗
輻射敏感性效果…………………………………………………....23
3.5 Galectin-1增強子宮頸癌細胞經輻射造成的DNA損傷
修補能力…………………………………………………………....24
3.6 調控ROGDI表現會影響各類腫瘤細胞生長速度………………..24
3.7 ROGDI在子宮頸癌細胞週期中扮演的角色……………………..25
3.8 降低ROGDI表現量會增加HeLa及C33A細胞對輻射的
敏感性……………………………………………………………....26
3.9 降低HeLa及C33A細胞ROGDI表現量會加速γ-H2AX (Ser139)
磷酸化表現………………………………………………………....27
第四章 討論…………………………………………………………….28
第五章 參考文獻……………………………………………………….33
Curriculum Vitae……………………………………………………….66

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