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研究生:許智傑
研究生(外文):Chih-Chieh
論文名稱:探討Epithelial Membrane Protein 2基因對人類肺癌細胞株生長與侵犯能力之影響
論文名稱(外文):Study the effect of epithelial membrane protein 2 gene on growth and invasion in human lung cancer cell lines
指導教授:蔡菁華
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:醫學分子毒理學研究所
學門:醫藥衛生學門
學類:其他醫藥衛生學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:72
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EMP2 (Epithelial membrane protein-2),為GAS3/PMP22 family的成員,以tetraspan形式表現在細胞膜上之蛋白質,已知可調控細胞生長、分化、細胞凋亡、以及影響細胞與細胞外基質蛋白質的相互作用。EMP2基因在肺癌病人之正常肺組織的表現量較肺腫瘤組織高約2~162倍。從MTT assay可知,當CL1-0細胞EMP2過度表現時,並不影響生長速率,而CL1-5細胞的生長速度有下降的趨勢。經由流式細胞儀分析細胞週期的分佈,發現控制組與實驗組並沒有太大差異。EMP2表現量增加時,在細胞正常貼附的狀況下(foci formation),不影響CL1-0細胞群落形成的能力,但CL1-5的細胞群落卻有變小的現象;而在細胞不貼附的狀態下(anchorage independent),EMP2過度表現可造成CL1-0以及CL1-5細胞形成細胞群落的能力下降。在沒有血清的狀況下,過度表現EMP2會引起CL1-0細胞的死亡。活體外試驗(in vitro)我們發現大量表現EMP2會降低CL1-5細胞的侵犯能力,更重要的是,將過度表現EMP2的CL1-5細胞經皮下注射至裸鼠中,沒有發現裸鼠有癌細胞轉移現象。由本研究結果顯示過度表現EMP2可以減緩細胞的生長,促進細胞在無血清狀態下的死亡,抑制腫瘤的侵犯,再加上先前在肺癌病人的組織中,偵測出EMP2在正常組織有較高的表現量,這些發現暗示著EMP2可能經過其抑癌基因的功能而影響肺癌細胞腫瘤形成的能力。

Epithelial membrane protein-2 (EMP2), a member of GAS3/PMP22 family, is a tetraspan protein and has been implicated in the modulation of cell growth, differentiation, cell apoptosis, and cell-extracellular matrix protein interactions. The mRNA level of EMP2 in normal lung was 2- to 162- fold higher than that in lung cancer. Overexpression of EMP2 in CL1-0 cells did not alter cell growth rate examined by MTT assay, but decrease the growth rate of CL1-5 cells compared with the control cells. There were no obvious differences in cell cycle distribution between parental cells and cells with overexpressed EMP2 examined by flow cytometry. Similar to MTS assay, increasing the expression of EMP2 decreased foci formation in CL1-5 cells, but no effect on foci formation in CL1-0 cells. However, overexpression of EMP2 decreased anchorage independent growth in both CL1-0 and CL1-5 cells. EMP2 induced CL1-0 cells death when serum was depleted from the culture medium. Elevation of EMP2 expression in CL1-5 cells reduced invasive ability in vitro. More importantly, when EMP2 overexpressed CL1-5 cells were injected subcutaneously into nude mice, none of the animals developed lung metastasis. In this study, overexpressed EMP2 restricted the cell growth, promoted cell death on serum deprivation and inhibited tumor invasion. All these findings together with the differential expression pattern of EMP2 in lung cancer patients suggest that EMP2 may influence lung tumorigenicity through a functional tumor suppressor phenotype.

壹、 中文摘要 1
貳、 英文摘要 2
參、 縮寫表 3
肆、 前言
一、 肺癌流行病學 4
二、 鑑定參與肺癌形成的基因 4
三、 人類EMP2基因 6
伍、 材料與儀器
一、 酵素 8
二、 商業套組 8
三、 試藥 8
四、 質體 9
五、 細胞株 9
六、 裸鼠品系 9
七、 儀器 9
陸、 實驗方法
一、 質體DNA的萃取 ( Plasmid extract ) 11
二、 建立穩定表現EMP2的細胞株 13
三、 肺癌細胞株培養 17
四、 篩選抗G418的Stable clone 19
五、 細胞染色體DNA的萃取 20
六、 細胞RNA的萃取 20
七、 利用去氧核糖核酸酶除去total RNA裡的染色體DNA 21
八、 反轉錄反應 ( Reverse Transcription;RT ) 22
九、 定量PCR(Real time PCR) 22
十、 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) 分析法 22
十一、 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) 分析法 23
十二、 計數細胞數目 (counting cell numbers) 23
十三、 流式細胞分析 (Flow cytometry) 24
十四、 細胞侵犯試驗 (Modified Boyden chamber) 25
十五、 群落形成試驗 (Colony formation) 25
十六、 軟培養基非附著性生長試驗 (Achorage independent assay) 26
十七、 細胞凋亡試驗 (Apoptosis assay) 26
十八、 活體腫瘤形成試驗 (Tumorigenicity assay) 27
十九、 統計分析 27
柒、 結果
一、 利用西方點墨法偵測穩定表現EMP2的細胞株 28
二、 利用RT-real time PCR定量細胞株中EMP2 mRNA過度表現的效果 29
三、 利用MTT assay觀察過度表現EMP2對CL1-0細胞生長的影響 30
四、 利用MTS assay觀察過度表現EMP2對CL1-5細胞生長的影響 31
五、 以流式細胞儀分析過度表現EMP2對細胞週期的影響 31
六、 利用細胞群落形成試驗(foci colony formation assay)觀察EMP2 overexpression對細胞群落形成的影響 32
七、 利用細胞不貼附試驗(anchorage independent assay)觀察EMP2過度表現對細胞群落形成的影響 33
八、 過度表現EMP2基因在無血清之壓力下對細胞凋亡的影響 35
九、 CL1-5細胞中EMP2基因過度表現對細胞侵犯能力的影響 35
十、 CL1-5細胞中EMP2基因過度表現對腫瘤形成的影響 36
捌、 討論
一、 N21與EMP2之間的關係 38
二、 過度表現的效果 39
三、 人類EMP2基因於癌症的角色 39
四、 抑癌基因的探討 40
五、 人類EMP2基因於生理功能的角色 44
玖、 圖目錄
圖一、 利用西方點墨法偵測穩定表現EMP2的CL1-0細胞株 46
圖二、 利用西方點墨法偵測穩定表現EMP2的CL1-5細胞株 47
圖三、 轉殖EMP2之CL1-0細胞株中,EMP2 mRNA過度表現的情形 48
圖四、 轉殖EMP2之CL1-5細胞株中,EMP2 mRNA過度表現的情形 49
圖五、 利用MTT assay 觀察EMP2 過度表現對CL1-0細胞生長的影響 50
圖六、 利用MTS assay 觀察EMP2 過度表現對CL1-5細胞生長的影響 51
圖七、 經流式細胞儀分析過度表現EMP2對CL1-0細胞週期的影響 52

圖八、 經流式細胞儀分析過度表現EMP2對CL1-5細胞週期的影響 53
圖九、 利用細胞群落形成試驗(foci colony formation assay)觀察EMP2過度表現對CL1-0細胞群落形成的影響 54
圖十、 利用細胞群落形成試驗(foci colony formation assay)觀察EMP2過度表現對CL1-5細胞群落形成的影響 55
圖十一、 利用細胞不貼附試驗(anchorage independent assay)
觀察EMP2 過度表現對CL1-0細胞群落形成的影響 56
圖十二、 利用細胞不貼附試驗(anchorage independent assay)觀察EMP2 過度表現對CL1-5細胞群落形成的影響 57
圖十三、 在細胞不貼附試驗(anchorage independent assay),CL1-5細胞中EMP2過度表現之細胞群落大於250 μm所佔之比例 58
圖十四、 經流式細胞儀分析過度表現EMP2在無血清的壓力下對CL1-0細胞週期的影響 59
圖十五、 經流式細胞儀分析過度表現EMP2在無血清的壓力下對CL1-5細胞週期的影響 60
圖十六、 CL1-5細胞中EMP2基因過度表現對細胞侵犯能力
的影響 61
圖十七、 CL1-5細胞中EMP2基因過度表現對腫瘤形成的影響 62
圖十八、 CL1-5細胞株所誘發的腫瘤形態 63
圖十九、 裸鼠腫瘤的生成率 64
拾、 表目錄
表一、 引子序列 65
表二、 過度表現EMP2的細胞株相對於18s rRNA表現量的相關性 66
拾壹、 附圖
附圖一、 EMP2過度表現質體的構築 68
拾貳、 參考文獻 69

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