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研究生(外文):Cai-hua Huang
論文名稱(外文):ROGDI is a novel positive cell cycle regulator
指導教授(外文):Cho, Chung-Lung
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ROGDI是一個功能未知的基因,位於人類染色體16p13.3上,遺傳編碼區有864 bp,轉譯出287個胺基酸。前人的研究指出過量表現ROGDI會促使數種細胞株增生。CDK2是一個會分別與cyclin E或cyclin A結合成複合體的細胞週期素依賴激酶,在G1/S過渡期扮演一個關鍵的調節角色,其活性從G1期一直持續到S期。在本研究中,我們發現CDK2和cyclin A的表現量會隨著過量表現ROGDI而增加;相反地,CDK2和cyclin A的表現量會隨著降表現ROGDI而減少。進一步利用免疫沈澱法證實ROGDI與CDK2在in vivo層級具有交互作用,同時透過免疫螢光染色可觀察到ROGDI與CDK2在細胞核有共位現象。我們利用olomoucine抑制CDK2活性,結果證實過量表現ROGDI促進細胞增生是會依賴CDK2的活性。藉由流式細胞儀分析顯示降表現ROGDI會造成細胞出現G1/S期停滯的現象,此時也偵測到p53與p21蛋白表現量增加。總結以上結果,我們推論ROGDI可能是一個依賴CDK2活性的細胞週期正向調節因子。
ROGDI is a novel gene which locates on human chromosome 16p13.3 and has unknown function. The length of ROGDI coding region is 864 bp which encodes 287 amino acids. According to our previous studies, overexpression of ROGDI increased proliferation in several human cell lines. CDK2 is a member of cyclin-dependent kinases (CDKs) which plays a key role in regulation of G1/S cell cycle transition and is activated during G1 and S phases. In this studies, we found that expression of CDK2 and cyclin A were up-regulated following overexpression of ROGDI in HEK293T. On the other hand, down-regulation of CDK2 and cyclin A was observed following silencing of ROGDI. In addition, the interaction between ROGDI and CDK2 was evidenced by using immunoprecipitation. Meanwhile, we observed co-localization of ROGDI and CDK2 in the nucleus. We also exploited a drug, olomoucine, known to selectively inhibit CDK2. The result demonstrated that ROGDI-enhanced cell proliferation was dependent on CDK2 activity. Flow cytometry profiles of depletion of ROGDI displayed G1/S phase arrest as well as up-reguated the expression of p53 and p21. Taken together, we suggest that ROGDI may be a positive regulator of cell cycle and its action is CDK2-depedent.
中文摘要 1
Abstract 2
目錄 3
壹、前言 5
二、細胞週期 (cell cycle) 6
三、細胞週期檢查點 (cell cycle checkpoints) 9
四、細胞週期素A/細胞週期素依賴性激酶2複合體 10
五、p53-p21反應途徑 (p53-p21 pathway) 11
貳、實驗目的 12
參、材料與方法 13
一、細菌培養 13
二、細胞培養 14
三、中量質體製備 15
四、Total RNA的萃取 17
五、反轉錄作用 18
六、聚合酶連鎖反應 19
七、西方墨點法 20
八、細胞質與細胞核的分離 26
九、真核細胞轉染 27
十、利用病毒產生RNA干擾 28
十一、免疫沈澱分析 30
十二、細胞週期同步化 31
十三、細胞生長曲線 33
十四、流式細胞儀分析 33
十五、免疫螢光染色 35
十六、數據統計分析 37
肆、結果 38
伍、討論 50
陸、參考文獻 58
柒、圖表 64
捌、附錄 88
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