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姓名	吳佳玲(Chia-Ling Wu)	電子郵件信箱	E-mail 資料不公開
畢業系所	生物科學系研究所(Biological Sciences)
畢業學位	碩士(Master)	畢業時期	92學年第2學期
論文名稱(中)	Dlk蛋白質的過度表現對人類肝癌細胞之癌症特性的影響
論文名稱(英)	The effect of Dlk overexpression on the tumorigenicity of hepatoma cells.
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		論文語文/頁數	中文/72
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摘要(中)	Dlk (delta-like)本身是一種跨膜蛋白質，構造上包含：細胞外區域、跨膜區域和細胞內區域。dlk的主要構造在於細胞外區域有六個重覆排列的類似表皮細胞生長因子。此外，在這段細胞外的構造上有一處可被酵素作用的切點，位置約接近細胞膜近側，在經不知名酵素作用後，會產生可溶片段並釋出細胞外去進行作用。dlk屬於EGF-like homeotic superfamily的成員之一，被認為在細胞分化過程中可能扮演重要角色。dlk在文獻中有一些不同的名稱，例如：人類腎上腺特有的pG2蛋白質、存在於胎兒羊水中的FA-1蛋白質、來自基質細胞的SCP-1蛋白質、表現於老鼠前脂肪細胞的Pref-1蛋白質以及老鼠腎小球區特有的ZOG蛋白質等，這些不同名稱的蛋白質其實是同一基因的多樣性產物。dlk的主要功能包括：抑制脂肪細胞形成、協助造血細胞分化成熟以及參與神經內分泌細胞的分化等。與dlk有關的腫瘤皆具神經內分泌的特性且似乎與腫瘤的分化階段有關，分化程度最差的腫瘤細胞並不會表現dlk。目前被發現會表現dlk的人類腫瘤則僅有神經母細胞瘤及肺小細胞癌。 Dlk會抑制脂肪細胞的形成，相對間接減少脂肪合成酶的產生。近年來陸續有文獻證實脂肪合成酶的大量表現與許多常見的癌症有關，因此dlk在調控脂肪細胞的同時可能也間接防止癌細胞產生。dlk與胎兒的分化有關，而某些幼兒的癌症是因分化失衡所造成，另外dlk亦被證實是肝臟開始纖維化的重要因子，而肝臟纖維化有時是肝癌的早期病癥，所以dlk與癌症的產生似乎有正向關係。 為進一步釐清dlk與腫瘤細胞之間的關聯性，實驗中讓惡性肝癌細胞 SK-Hep-1過度表現dlk蛋白質，藉此來觀察分析細胞的癌症特色是否有所改變。實驗中針對dlk的全長及extracellular domain等不同蛋白質片段來進行分析，結果發現在缺乏血清的環境中，表現extracellular domain的細胞株均呈穩定生長，經細胞週期分析後發現其細胞凋亡的現象遠比其他細胞株輕微。將各細胞株以皮下注射殖入SCID mice體內，結果以攜帶dlk全長的細胞株所引發的腫瘤生長最為迅速，腫瘤體積最大。利用Boyden chambers及人為裂隙(gap)等方法進行細胞移行能力的分析，結果發現表現dlk的細胞株，不論是全長或extracellular domain，其移行能力均較對照組明顯減弱。因此，綜觀以上現象，dlk全長基因可以促進老鼠腫瘤的生長，而其細胞外的EGF-like repeats，具生長因子及抗細胞凋亡功能，可以協助細胞在缺乏血清的環境中穩定生長。
摘要(英)	Dlk is a transmembrane protein that possesses six epidermal growth factor-like sequences at the extracellular domain, a single transmembrane domain and an intracellular tail. The extracellular EFG-like region of Dlk can be released by action of an unknown protease that cuts the extracellular region near the cell membrane. Dlk belongs to the EGF-like homeotic protein family and has received many names: pG2, FA-1, Pref-1, SCP-1, ZOG and Dlk. All the proteins are identical or polymorphic products of a single gene. Dlk has been involved in several differentiation processes, such as adipogenesis, hematopoiesis and neuroendocrine differentiation. Dlk is also known as the preadipocyte factor-1 (Pref-1), is highly expressed in preadipocytes but is completely abolished in adipocytes. Pref-1 may function in the maintenance of the preadipocyte state and is a negative regulator of adipocyte differentiation. Dlk is expressed in tumors with neuroendocrine features, such as human neuroblastoma, rat pheochromocytoma, and a subset of Small Cell Lung Cancer (SCLC) cell lines. The Dlk expression is probably associated with some differentiation stages because the most undifferentiated cells were lacking expression of Dlk. The finding suggests that Dlk plays an important role in differentiation and tumorigenesis of several cell types. The study was designed to examine the influence of dlk overexpression on tumorigenicity of hepatoma cells. We constructed the mammalian expression vectors for full-length dlk, dlk extracellular domain, which were transfected into SK-Hep-1 cells for generation of stable clones. The transgene expressions in selected stable clones were verified by QRT-PCR and western blot analysis. Our results indicated that overexpression of extracellular domain significantly promoted the viability of SK-Hep1 cells during serum deprivation. In SCID mice, injection of full-length dlk clones led to increased tumor growth compared with the control groups. However, the migration ability was reduced in Dlk stable clones. In summary, these results suggested full-length Dlk promoted the tumor growth but reduced the migration ability of SK-Hep1 cells.
關鍵字(中)	Dlk蛋白質 過度表現 肝癌細胞 癌症特性	關鍵字(英)	overexpression tumorigenicity hepatoma cells Dlk protein
論文目次	目錄 目錄 …………………………………………………… 1 中文摘要 ……………………………………………… 6 英文摘要 ……………………………………………… 8 縮寫 ………………………………………………… 10 前言 …………………………………………………… 11 實驗背景 ……………………………………………… 17 實驗目的 ……………………………………………… 18 實驗方法與材料 ……………………………………… 19  細胞株(Cell line) …………………………………… 19  細胞培養基(Culture medium) …………………… 19             細胞培養(Cell culture) …………………………… 19  抽取total RNA …………………………………… 20  製備cDNA(RT-PCR) ……………………………… 20  聚合酶連鎖反應(PCR) …………………………… 21  DNA電泳…………………………………………… 22  純化PCR產物 ……………………………………… 23  phCMV3載體的黏合反應(Ligation) …………… 23  轉形作用(Transformation) ……………………… 23  含轉形基因菌體的篩選 …………………………… 24  純化質體DNA ……………………………………… 24  核酸定序 …………………………………………… 25  細胞數目的測定 …………………………………… 26  轉染(Transfection)與選殖表現dlk基因的 細胞株 ……………………………………………… 27  選殖穩定表現dlk基因的細胞株(Selection of stable clone) ………………………………………… 28  蛋白質濃度分析 …………………………………… 28  SDS-聚丙烯醯胺膠體電泳法(SDS-polyacrylamide  gel electrophoresis) ………………………………… 29  西方墨點法(Western blotting) …………………… 30  即時定量聚合酶連鎖反應(Real-time PCR) ……… 31  生長曲線(Growth curve) ………………………… 32  細胞移行分析(Chemotaxis assay) ………………… 32  細胞週期的分析(Cell cycle analysis) …………… 33  體內試驗(In vivo) ………………………………… 34 結果 …………………………………………………… 35  轉染SK-Hep-1並選殖出穩定且過度表現dlk 蛋白質的細胞株 …………………………………… 35    比較各細胞株脂肪合成酶(FAS)的表現量 …… 39   比較各細胞株的生長型態(Cell morphology) …… 40  生長曲線分析 ……………………………………… 41  細胞週期分析 ……………………………………… 41  細胞移行分析 ……………………………………… 42  體內試驗 …………………………………………… 43  以西方墨點法再次確認各細胞株所攜帶 的基因 ……………………………………………… 43 討論 …………………………………………………… 45 參考文獻 ……………………………………………… 48 圖表 …………………………………………………… 52  圖1. dlk蛋白質的構造 …………………………… 52  圖2. 不同的人類肝癌細胞其dlk基因表現有 明顯差別 …………………………………………… 53    圖3. 以Western blot鑑定攜帶dlk或GFP 基因的細胞株 ……………………………………… 54  圖4. 以real-time PCR確認攜帶dlk細胞外 基因的細胞株 ……………………………………… 55  圖5. 以Western blot比較各細胞株的脂肪 合成酶(FAS)表現量 ……………………………… 56            圖6. 各細胞株的細胞型態 ……………………… 57  圖7. 細胞株GFP(1)與GFP(2)在螢光顯微鏡下 所呈現的綠螢光情形 ……………………………… 58  圖8. 細胞株在不含血清的環境下所呈現之 生長曲線 …………………………………………… 59 圖9. 細胞株在含10﹪血清的環境下所呈現 之生長曲線 ……………………………………… 60  圖10. 細胞在無血清狀態下培養24小時， 之後利用流式細胞儀分析其細胞週期 …………… 61  圖11. 細胞在無血清狀態下培養24小時， 之後利用流式細胞儀分析其細胞週期 …………… 62  圖12. 細胞在無血清狀態下培養48小時， 之後利用流式細胞儀分析其細胞週期 …………… 63  圖13. 細胞在10﹪血清狀態下培養24小時， 之後利用流式細胞儀分析其細胞週期 …………… 64  圖14. 細胞在10﹪血清狀態下培養48小時， 之後利用流式細胞儀分析其細胞週期 …………… 65  圖15. SK-Hep-1, EC(1), EC(5)與Dlk(3)等 細胞株在通過濾膜後被染色的情形 ……………… 66  圖16. 細胞株移行(migration)能力的比較 ……… 67    圖17. 細胞株填補空隙(gap)能力的比較 ………… 68  圖18. 各細胞株在SCID mice體內引發腫瘤 生長的情形 ………………………………………… 69  圖19. 以Western blot再次確認各細胞株所 表現的蛋白質 ……………………………………… 70 附圖 …………………………………………………… 71  載體phCMV3的圖譜 ……………………………… 71
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口試委員	陳錦翠 - 召集委員 卓忠隆 - 委員 戴明泓 - 指導教授 莊錦豪 - 指導教授
口試日期	2004-06-28	繳交日期	2004-09-04

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