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研究生:許博彥
研究生(外文):Po Yen Hsu
論文名稱:人類肝癌大量表現基因-FLJ11252之功能性研究
論文名稱(外文):Functional Characterization of a Human HCC Highly Expressed Novel Gene-FLJ11252
指導教授:周成功
指導教授(外文):C. K. Chou
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
論文頁數:58
中文關鍵詞:肝癌細胞週期RhoGAP
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本實驗室藉由分析Stanford cDNA microarray資料庫,篩選出一群在肝癌細胞大量表現,且表現量會隨著細胞週期變化的基因。本研究選擇FLJ11252這個未知功能的新穎基因進行研究,以期瞭解這個基因在肝癌細胞生長調控上所扮演的角色
FLJ11252位於人類第五號染色體上,具有一段類似RhoGAP domain的保留性功能區域。先前的研究證實FLJ11252 mRNA在G2/M時期會被大量表現。在肝癌病人的肝癌細胞組織中,FLJ11252的表現量被大大提升。在細胞中過量表現FLJ11252時,則會讓細胞停留在G2/M時期。
利用純化後的FLJ11252 蛋白所產生的多株抗體去進行西方點墨法分析,確認了FLJ11252在肝癌細胞株中的表現量,FLJ11252的蛋白質與mRNA在HeLa細胞中會在G2/M時期大量提升,進入 G1時期後慢慢減少。進一步利用RNAi將細胞內FLJ11252 的表現抑制後發現,細胞的生長速率變慢且細胞位於G1時期的比例增加,ERK被磷酸化的現象也跟著減少。藉由pull-down及免疫沈澱試驗發現FLJ11252會與細胞內的Cdc20及MEK2相互結合,推測FLJ11252藉由與這兩個蛋白的結合,分別調控細胞分裂時期的進行及影響細胞的生長速率。最後我發現,抑制FLJ11252的表現後,細胞移動性及侵入性都會降低,推測FLJ11252可能是一個RhoGAP的negative dominant regulator,可以透過調控細胞內Rho GTPases的活性來刺激細胞的移動力。
Hepatocellular carcinoma is one of the most frequent malignant neoplasm in Taiwan. Therefore, investigating the cell cycle regulatory genes and analysis their functions in the carcinoma cells might shed a new light for better understanding and treatments of hepatocellular carcinoma.
Bioinformatics analysis of microarray database has been performed to identify genes are upregulated in human heptocellular carcinoma (HCC) tissues and are also under cell cycle regulation. One novel gene FLJ11252 was shown overexpressed in human HCC tissues and induced to express during G2/M phase of regenerating mouse liver. Ectopic expression of FLJ11252 in 293T cells blocks cell cycle progression at G2/M phase. Structure analysis reveals that FLJ11252 contains an incomplete RhoGAP domain. Whether this incomplete RhoGAP domain makes FLJ11252 as a negative dominant regulator of normal RhoGAP and to keep small G protein active is currently under investigation.
We have shown that FLJ11252 is a cell cycle regulated protein, which up-regulated in G2/M phase and localized on the cytosol and cell membrane. When FLJ11252 was knockdowned by interference RNA in human HCC cells, we observed that the cell growth was slow down and arrested in G1 phase. Moreover, cell invasion activity was also been suppressed in the FLJ11252 knockdowned HCC cells. Finally, FLJ11252 physically interacts with Cdc20 and MEK2 respectively. Such interactions may explain the functional role of FLJ11252 in both G2/M and G1 transition of cell cycle in HCC cells.
目錄

指導教授推薦書……………………………………………………… i
口試委員會審定書…………………………………………………… ii
授權書………………………………………………………………… iii
誌謝…………………………………………………………………… iv
中文摘要………………………………………………………………… v
英文摘要……………………………………………………………… vi
目錄…………………………………………………………………… vii
圖表目錄………………………………………………………………… x
第一章、 緒論 ………………………………………………………… 1
1.1 肝癌的生成的原因………………………………………… 1
1.2 細胞週期的變異與癌症的相互關係................ 2
1.3 運用生物資訊學的方法找尋肝癌相關基因........... 3
1.4 FLJ11252的相關認知…………………………………… 4
1.5 Rho GTPases與RhoGAPs ……………………………… 5
1.6 Rho蛋白與細胞的移動性…………………………………… 6
1.7 研究目的…………………………………………………… 7
第二章、 實驗材料與方法 ………………………………………… 8
2.1 細胞培養…………………………………………………… 8
2.2 細胞週期之同化…………………………………………… 8
2.3 流式細胞儀分析…………………………………………… 8
2.4 細胞數量之計數…………………………………………… 9
2.5 質體之建構………………………………………………… 9
2.6 質體之製備………………………………………………… 10
2.7 細菌的轉型作用…………………………………………… 10
2.8 細胞total RNA之抽取……………………………………… 11
2.9 反轉錄聚合脢連鎖反應…………………………………… 12
2.10 DNA的轉染………………………………………………… 13
2.11 細胞蛋白質之粹取………………………………………… 13
2.12 西方點墨法……………………………………………… 14
2.13 His-FLJ11252融合蛋白的誘導與純化………………… 15
2.14 FLJ11252多株抗體之純化………………………………… 16
2.15 利用慢病毒之DNA轉染法………………………………… 16
2.16 篩選抑制FLJ11252表現之細胞株………………………… 17
2.17 免疫螢光染色法…………………………………………… 17
2.18 沉澱試驗…………………………………………………… 18
2.19 免疫共沈澱法……………………………………………… 18
2.20 免疫沈澱法….……………………………………………… 19
2.21 細胞移動性試驗…………………………………………… 19
2.22 細胞侵入性試驗…………………………………………… 20
第三章、 結果 ……………………………………………………… 22
3.1 FLJ11252多株抗體之專一性測試………………………… 22
3.2 不同細胞株內生性FLJ11252的表現量…………………… 22
3.3 FLJ11252在細胞株表現的位置…………………………… 23
3.4 FLJ11252的表現會隨著細胞週期的進行而跟著改變…… 23
3.5 FLJ11252 RNAi具有抑制內生性FLJ11252表現的能力…… 24
3.6 抑制細胞內FLJ11252導致肝癌細胞株生長速度減慢,
並讓細胞G1時期比例增加……………………………………… 24
3.7 抑制掉細胞內FLJ11252對細胞訊息傳遞作用的影響…… 25
3.8 抑制細胞內FLJ11252的表現使細胞移動及侵入性降低… 26
3.9 FLJ11252會跟細胞內的Cdc20及MEK2進行結合 ………… 26
第四章、 討論 …………………………………………………………… 28
4.1 細胞內FLJ11252的表現對細胞週期造成的影響………… 28
4.2 FLJ11252對細胞內訊息傳遞作用影響…………………… 29
4.3 FLJ11252對細胞內Rho GTPase活性的影響……………… 31
4.4 FLJ11252可能是一個致癌基因………………………… 32
第五章、 圖表 ………………………………………………………… 33
參考文獻 …………………………………………………………… 45



圖表目錄

圖(一)、FLJ11252多株抗體可專一的辨認到細胞內FLJ11252蛋
白質.......................................... 33
圖(二)、FLJ11252多株抗體可專一性的辨認到細胞內N端及C端
的FLJ11252蛋白................................ 34
圖(三)、外生性FLJ11252表現在細胞質及細胞膜上............ 35
圖(四)、細胞內FLJ11252表現受到細胞週期的調控而改變………… 36
圖(五)、FLJ11252 RNAi具有抑制FLJ11252在細胞內表現能力… 37
圖(六)、FLJ11252被抑制的HuH-7細胞生長速率變慢,且會抑制
ERK的活性………………………………………………………38
圖(七)、FLJ11252被抑制的HepG2細胞生長速率變慢,且會抑制
ERK的活性,並使細胞G1時期的比例增加………………… 40
圖(八)、抑制FLJ11252的表現,導致細胞移動性及侵入性降低… 42
圖(九)、FLJ11252會與細胞內MEK2及Cdc20進行交互作用…… 44
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