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博碩士論文 etd-0829105-122014 詳細資訊
Title page for etd-0829105-122014
論文名稱
Title
血管新生抑制劑vasostatin基因傳送在癌症治療之研究
Gene Delivery of Angiogenesis Inhibitor Vasostatin for Cancer Therapy
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
58
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
2005-07-28
繳交日期
Date of Submission
2005-08-29
關鍵字
Keywords
內皮細胞、腫瘤、線病毒、血管新生
adenovirus, vasostatin, tumor, angiogenesis, endothelial cell
統計
Statistics
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中文摘要
腫瘤的生長和轉移必須依靠血管新生。 Vasostatin為一種內生性血管新生抑制劑,是calreticulin水解切出的N端180個胺基酸。研究指出vasostatin可專一性地抑制內皮細胞增生與腫瘤生長。 然而,持續性給予vasostatin不僅困難而且昂貴。 因此,希望發展基因傳送的方式。 腺病毒載體具有高力價、高感染效率,及廣泛寄主等優點。 本實驗目的在於產生表現vasostatin (Ad-VS) 與接有Igk 之vasostatin重組腺病毒(Ad-Igk-VS),進而評估其抗血管新生基因治療對抑制腫瘤的效果。 利用聚合
Abstract
The growth and metastasis of solid tumors are dependent on angiogenesis. An endogenous angiogenesis inhibitor, vasostatin, is the proteolytic fragment derived from the N-terminal 180 residues of calreticulin. Previous studies indicated that vasostatin specifically inhibits endothelial cell proliferation, angiogenesis and tumor growth. However, continuous administration of vasostatin is difficult and expensive to facilitate, thereby underscoring the need to develop gene delivery approach. Adenovirus vectors possess advantages for gene delivery including high titer, high infection efficiency and broad host range. The aim of the present study was to generate and characterize recombinant adenovirus vectors encoding vasostatin (Ad-VS) or Igk-fused vasostatin (Ad-Igk-VS), thereby to evaluate the efficacy of anti-angiogenesis gene therapy for tumor suppression. Recombinant Ad-VS and Ad-Igk-VS were generated and verified by PCR and western blot analysis. In addition, adenovirus encoding angiostatin was also produced as positive control for angiogenesis assays. Adenovirus-mediated vasostatin gene delivery specifically inhibited the proliferation of bovine aortic endothelial cells (BAEC), but not non-endothelial cells such as Hela or NIH3T3 cells. Moreover, vasostatin gene delivery potently inhibited the proliferation, migration and tube formation, but not secretion of matrix metalloproteinases (MMPs), in endothelial cells. Flow cytometry analysis indicated that vasostatin gene delivery induced apoptosis in BAEC. Using western blot analysis, it was revealed that gene delivery of vasostatin increased the levels of Fas and FADD in BAEC. In conclusion, adenovirus-mediated vasostatin gene delivery inhibited various angiogenesis processes at least via induction of Fas/FasL pathway and may hold potential for cancer therapy.
目次 Table of Contents
Contents…………………………………………………………Ⅰ
Abstract in Chinese………………………………………………………………. Ⅳ
Abstract in English……………………………………………………………........Ⅴ
Abbreviations…………………………………………Ⅵ

Introduction
Angiogenesis...................................1
Angiogenesis activator and inhibitor......................................2
Angiogenesis inhibitor and vasostatin……………………………………………….2
Gene delivery and adenovirus vectors………………………………………………. 4
Specific aims……………………………………………6

Materials and Methods
Cell cultures……………………………………………7
Western blot analysis……………………………………………………7
PCR analysis………………………………………………8
Generation of adenovirus encoding vasostatin (Ad –VS and Ad-Igk-VS)……….. 8
Amplification and purification of adenovirus vectors…………………………….. 9
Determination of optimal MOI for adenovirus to infect cells……………………. .9
Adenovirus gene delivery……………………………………………………………10
Cell proliferation assay………………………………………………………………10
Cell migration assay……………………………………………………………….11
Gelatine zymography…………………………………………………………11
Tube formation assay…………………………………………………………………12
Flow cytometry assay…………………………………………………………………13
Statistic analysis……………………………………………………………13

Results
Generation and verification of recombinant adenovirus encoding vasostatin
(Ad-VS and Ad-Igk-VS)…………………………………………………..14
Adenovirus-mediated vasostatin gene delivery led to vasostatin release in
infected cells……………………………………………………………………………. 14
Vasostatin gene delivery inhibited the proliferation of endothelial cells…………… 15
Vasostatin gene delivery attenuated the migration of endothelial cells…………….. 15
Vasostatin gene delivery reduce tube formation of BAEC…………………………... 16
Gene delivery of vasostatin had no significant effect on MMP-2 and –9 secretion in endothelial cells…………………………………………………………16
Gene delivery of vasostatin induced apoptosis in BAEC cells……………………….17
Gene delivery of vasostatin increased the level of Fas and FAAD in BAEC cells......17

Discussion……………………………………………………18

Future perspectives…………………………………………21

References………………………………………………………23

Tables…………………………………………………………… 27

Figures and Legends……………………………………………………………29

Appendix…………………………………………………………43
參考文獻 References
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