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系統識別號 U0026-0812200915353908
論文名稱(中文) 慢病毒載體攜帶血管內皮細胞生長因子之基因導入促進外科皮瓣存活與血管新生
論文名稱(英文) Lentivirus-Mediated Gene Transfer of Vascular Endothelial Growth Factor Improves Surgical Flap Survival and Angiogenesis
校院名稱 成功大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Institute of Clinical Medicine
學年度 97
學期 2
出版年 98
研究生(中文) 蔡文平
研究生(英文) Wen-ping Tsai
學號 s9695102
學位類別 碩士
語文別 英文
論文頁數 43頁
口試委員 指導教授-蕭璦莉
口試委員-吳昭良
口試委員-吳梨華
指導教授-李經維
中文關鍵字 基因治療  慢病毒  血管內皮細胞生長因子  手術延遲 
英文關鍵字 surgical delay  VEGF  lentivirus  Gene therapy 
學科別分類
中文摘要 整形重建手術的領域中,表皮或肌肉皮瓣的缺血壞死是一個重大的臨床併發症,且缺乏有效處理治療方式。自很久以來,˝手術延遲˝這個技巧就已經開始被應用,使重建皮辦的存活更為可靠。為了避免額外的再次手術,我們一直持續尋找有效的˝非手術延遲˝方式。而其中一個具有發展性的方式即為基因治療。過去曾有人發表以腺病毒載體(adenoviral vector)及腺病毒相關載體(adeno-associated virus, AAV)等病毒載體攜帶血管新生基因,證實能有效促進皮瓣血管新生,但目前為止尚未有人利用到慢病毒(lentivirus)載體於相關研究。慢病毒載體有別於其他載體的優勢在於低宿主免疫反應、長時間的表現、以及很廣泛的宿主驅性包括不分裂的細胞等。從以上的觀察,我們假設慢病毒媒介之血管內皮細胞生長因子(vascular endothelial growth factor, VEGF)基因導入,能模擬手術延遲的效果於大鼠之橫腹直肌皮瓣(transverse rectus abdominis musculocutaneous, TRAM)動物模型。於此研究中,首先先構築攜帶血管內皮細胞生長因子的慢病毒載體,藉由促進內皮細胞的增生與移動來證實其活體外的血管新生能力。接著我們應用此病毒載體證實可模擬手術延遲,以促進皮瓣存活及增進血管新生於此一皮辦動物模式。最後也發現了出病毒載體劑量與效果的關係,以及可能的長期效應。此研究的結果,增加了應用慢病毒載體媒介之治療性血管新生於未來臨床重建手術之可能性。
英文摘要 In the field of plastic surgery, ischemic necrosis of skin or muscle flaps presents a major clinical problem in patients undergoing reconstructive procedures, with significant morbidity and no effective therapy available. The technique of “surgical delay” has long been well-used for making the survival of flap more reliable. In order to avoid additional surgery, effective “non-surgical delay” alternatives are constantly sought. One of the promising modalities is gene therapy. Several virus-mediated angiogenic gene transfer for enhancing flap survival have been proved to be beneficial, including adenovirus and adeno-associated virus (AAV) but not lentivirus (Lv). Lentivirus has several advantages than other vectors including low immunogenity, long-time expression, and broad tropism including non-dividing cell. From the above observations, we hypothesized that Lv-mediated vascular endothelial growth factor (VEGF) gene transfer can mimic surgical delay in the rat transverse rectus abdominis musculocutaneous (TRAM) flap animal model. In this study, we first constructed Lv-VEGF, a Lv-expressing VEGF, and verified its angiogenic effect by enhancing both migration and proliferation of endothelial cells. In the rat TRAM flap model, we were able to mimic surgical delay by introducing Lt-VEGF in enhancing flap survival and neovascularization. Dose-dependent effect and possible long-term effect of Lv-VEGF were also demonstrated in our TRAM flap model. The results of this study raised the possibility of using lentivirus-mediated therapeutic angiogenesis for future clinical use in reconstructive surgery.
論文目次 Abstract I
中文摘要 III
Acknowledgement V
Table of content VI
List of tables VIII
List of figures IX
Abbreviations X

Introduction 1
VEGF 3
Lentiviral vector 5
Aim of the study 6
Material and Methods 7
Materials 7
Plasmids 7
Cell lines 7
Animals 8
Methods 8
Cell lines and cell culture 8
Lv-VEGF construction 8
Production of lentivirus 9
Determination of viral titer 9
Preparation of conditioned medium 10
Infection efficiency of lentivirus to endothelial cells 10
Expression of the cloned VEGF gene 10
Cell Proliferation Assay 11
Cell Migration Assay11
TRAM flap animal model and the delay procedure 12
Flap tissue histology 13
Immunohistochemistry 14
Dose-dependent effect of Lv-VEGF 14
Long term effect of Lv-VEGF 15
Statistical analysis 15
Results 16
Construction of pWPI-VEGF 16
The in vitro effect of Lv-VEGF to endothelial cells 16
In vitro effect of Lv-VEGF to endothelial cells 16
Lv-VEGF improved flap survival by enhancing angiogenesis in TRAM flap model 17
Dose-dependent and long term effect of Lv-VEGF 19
Discussion 20
Reference 26
Table 33
Figures 34
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