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博碩士論文 etd-0822107-141847 詳細資訊
Title page for etd-0822107-141847
論文名稱
Title
探討Delta-Like 1 Homologue蛋白之胞外區在內皮細胞的血管新生功能與訊息傳遞路徑
The Angiogenic Functions and Signaling of Delta-Like 1 Homologue Extracellular Domain in Endothelial Cells
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
63
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2007-07-11
繳交日期
Date of Submission
2007-08-22
關鍵字
Keywords
血管新生
p38 MAPK, signaling pathway, ERK, eNOS, Akt, Hes1, Notch1, tube formation, migration, endothelial cell, DLK1
統計
Statistics
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中文摘要
Delta-like1 Homologue 簡稱DLK1,為一具有383個胺基酸的跨膜蛋白質,屬於含有像表皮生長因子(EGF)重複序列的家族,此家族中含有與決定細胞命運有關的Notch、Delta和Serrate。 DLK1又稱為preadipocyte factor-1 (簡稱Pref-1) ,因為基因多型性產物而有許多別名如: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, 表現於老鼠前脂肪細胞的Pref-1蛋白質。 DLK1細胞外區域 (extracellular domain of DLK1; DLK1-EC) 含有六個EGF重複序列並可經由tumor necrosis factor alpha converting enzyme (TACE) 切割釋放進行作用。 DLK1在細胞分化中可能扮演細胞重要角色,包含脂肪新生、造血分化和腎上腺分化有關的過程。 除此之外,在膽道閉鎖與神經膠質瘤的病人當中,DLK1會有過度表現的現象。最近,發現一重組蛋白thrombomodulin的細胞外區域含有六個EGF的重複序列且在體內與體外證實可以促進血管新生。所以我們進一步假設DLK1-EC除了可以調控脂肪新生外,也可能在血管新生中扮演一定的角色。為了驗證這個假設,在我的研究計劃中,首先利用大腸桿菌表現系統生產DLK1細胞外區域的蛋白質。為了測試重組蛋白在脂肪新生的生物活性,將DLK1細胞外區域加入人類的脂肪幹細胞,以確認他的生物功能。最後,DLK1-EC分別在體內與體外血管新生的活性測試,雖然DLK1-EC對matrix-metalloproteinase (MMPs)沒有什麼影響,外加的DLK1-EC在內皮細胞會顯著促進內皮細胞增生、移行能力與生成微血管狀的能力。另外,將DLK1-EC植入大鼠的角膜周邊會有誘導劑量效應的血管新生。綜而言之,DLK1經由酵素切割後所產生的DLK1-EC在體內與體外證實可以促進血管新生。除此之外,我們也嘗試在體外尋找DLK1可能透過何路徑來影響血管新生。而西方墨點法結果也證實,外加的DLK1-EC可能透過Notch1使Hes1活化下游的訊息傳遞路徑如 Akt/eNOS, p38 MAPK, ERK來達成其促進血管新生的功能。而之後利用Notch-1訊息傳遞路徑的抑制劑DAPT會抑制因DLK1-EC促進血管新生的效應。而本實驗指出DLK1-EC活化Notch-1主要是透過γ-secretase並活化下游的Akt/eNOS,所以Notch1也被認為在DLK1-EC誘導的血管新生中扮演重要的角色。未來我們希望這些研究可以進一步的了解DLK1的生理與病理的機轉。
Abstract
Delta-like 1 Homologue (DLK1), a transmembrane protein of 383 amino acids, belongs to a family of epidermal growth factor (EGF)-like repeat-containing proteins that include Notch/Delta/Serrate, which are involved in cell fate determination. DLK1 is also known as preadipocyte factor-1, pG2, and FA-1, which are identical or polymorphic products of a single gene. Structural analysis revealed that DLK1 consists of an extracellular domain with six EGF-like repeats, a transmembrane domain, and an intracellular domain. The extracellular EGF-like region of DLK1 (DLK1-EC) can be released to the medium by the action of tumor necrosis factor alpha converting enzyme (TACE). DLK1 participates in various differentiation processes including adipogenesis, hematopoiesis, and adrenal gland differentiation. Besides, DLK1 overexpression was observed in patients with biliary atresia and in glioblastoma. Recently, the extracellular domain of thrombomodulin, which also contains six EGF–like structures, has been delineated to stimulate angiogenesis in vitro and in vivo. This prompted us to investigate whether DLK1-EC played a role in angiogenesis. To test such hypothesis, recombinant DLK1-EC was expressed and purified in E. coli. Adding DLK1-EC recombinant protein inhibited the adipogenesis of adipocytes-derived stem cells in a dose-dependent manner. Despite marginal effect on matrix-metalloproteinase secretion, exogenous DLK1-EC significantly stimulated the proliferation, motility and tube-forming capability of cultured endothelial cells. Above all, implantation of DLK1-EC-containing hydron pellets induced cornea neovascularization in a dose-dependent manner. Western blot analysis revealed that exogenous DLK1-EC induced angiogenesis through Notch1 activating downstream gene Hes1 and subsequently signaling such as Akt/eNOS, p38 MAPK, and ERK pathway to perform its function. Indeed, blockade of Notch1 signaling using γ-secretase inhibitor leads to decreased angiogenesis and inhibits DLK1 EC-induced
endothelial cell tubular formation in vitro and in vivo. These findings indicate that
DLK1-EC induced Notch1 activation mediated by γ-secretase and tansactivation
Akt/eNOS pathway and that Notch1 is critical for DLK1 EC-induced angiogenesis.
These results may bring further insights into the physiological and pathological
functions of DLK1
目次 Table of Contents
Abstract in Chinese---------------------------------------------------I
Abstract in English-------------------------------------------------III
Contents
Introduction------------------------------------------------------------1
Angiogenesis-----------------------------------------------------------1
Delta-like 1 Homologue (DLK1)--------------------------------------------2
The Notch Signaling Pathway-----------------------------------------------4
Notch and DLK1 in angiogenesis ------------------------------------------5
Specific Aims-- --------------------------------------------------------7
Material and Methods-----------------------------------------------8
Expression vectors-------------------------------------------------------------8
Generation of DLK1 recombinant proteins-------------------------------8
Cell Culture------------------------------------------------------------8
Western blotting analysis-----------------------------------------------------9
Proliferation assay-----------------------------------------------------------10
Boyden chamber migration assay-----------------------------------------11
Tube formation---------------------------------------------------------------11
Adipogenesis assay----------------------------------------------------------11
Gelatin zymography assay-------------------------------------------------12
Corneal angiogenesis assay-----------------------------------------------13
Aortic ring assay-------------------------------------------------------------13
Statistic analysis-------------------------------------------------------------14
Results-----------------------------------------------------------------14
Expression and purification of 6xHis-fused DLK1-EC --------------15
Application of recombinant DLK1-EC attenuated the adipogenesis of adipocytes-derived stem cells in a dose-dependent manner ----------15
Recombinant DLK1-EC enhanced the proliferation of endothelial cells-----------------------------------------------------------------------------15
Recombinant DLK1-EC stimulated the migration of endothelial cells-----------------------------------------------------------------------------16
Recombinant DLK1-EC promoted the tube formation of endothelial cells-----------------------------------------------------------------------------16
DLK1-EC had no effect on MMP-2 and MMP-9 secretion in endothelial cells--------------------------------------------------------------17
Implantation of pellets containing DLK1-EC induced cornea angiogenesis------------------------------------------------------------------17
DLK1-EC induces activation of Notch1, Hes1, Akt/eNOS, ERK1/2 and p38 MAPK in endothelial cells --------------------------------------17
DLK1 EC-induced Notch1 cleavage and Hes1 expression mediate downstream signaling: Akt/eNOS, p38 MAPK and ERK activation18
Blockade of Notch1 activation abolished the DLK1-EC-induced tube formation and vessels sprouting ------------------------------------------18
Discussion-------------------------------------------------------------20
Future Perspectives-------------------------------------------------25
References------------------------------------------------------------26
Figures and Legends-----------------------------------------------31
Appendix-------------------------------------------------------------50
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