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系統識別號 U0026-1007201013042100
論文名稱(中文) 轉殖血管內皮生長因子之內皮幹細胞在下肢缺血小鼠模式中可提高治療性血管新生能力
論文名稱(英文) Transplantation of Endothelial Progenitor Cells Transduced with Vascular Endothelial Growth Factor Augments Therapeutic Neovasculization in a Mouse Hindlimb Ischemia Model
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 98
學期 2
出版年 99
研究生(中文) 陳倩怡
研究生(英文) Chian-Yi Chen
學號 S4696110
學位類別 碩士
語文別 中文
論文頁數 52頁
口試委員 指導教授-蕭璦莉
口試委員-吳昭良
口試委員-蔡文展
中文關鍵字 內皮幹細胞  慢病毒  轉染  缺血  血管新生  血管內皮生長因子 
英文關鍵字 EPC  lentiviral  transduced  ischemia  angiogenesis  VEGF 
學科別分類
中文摘要 缺血性心血管疾病是目前全球疾病中死因的主因。為了治療這些相關的症狀,除非有足夠的修復機轉,才能使內皮細胞層恢復並維持血管的恆常性。自從endothelial progenitor cell (EPC) 在周邊血液被發現以後,許多研究者使用各種不同的方法來分離及證實這群細胞。許多研究證明EPC有兩種明顯不同的遺傳表型,也就是 early (outgrowth) EPC及late (outgrowth) EPC。late EPC在這個系統中是最有分化能力的先驅細胞。它雖然能夠獨立形成血管,但卻沒有旁泌素等作用在分泌細胞鄰近細胞的作用。相反地,early EPC會表現泛白血球標記CD45以及單核白血球標記CD14,但卻只有低的增生能力。我們使用人類臍帶血,並從其中單核球細胞中分離出內皮幹細胞 (late EPC)。近年來的研究指出,臍帶血已成為一項十分常見的EPC分離方式,因為它比起成人的周邊血具有更多的EPC包含在其中。我們選用了長久以來被研究的十分透徹且在血管新生過程十分重要的血管內皮生長因子(vascular endothelial growth factor,VEGF)來做為一個轉殖的因子。在現今一些已知促進血管生長的因子之中,VEGF是目前最有影響力的血管新生因子,它除了參與了重要的一些血管新生步驟之外,特別在使EPC在血流中的移動,也扮演了重要的角色。late EPC經繼代培養擴增後,我們構築了表現VEGF慢病毒質體pWPI-vegf165及製備了慢病毒顆粒。接著以流式細胞儀測定綠色螢光,證實90%以上之late EPC轉殖VEGF成功。在細胞實驗方面,單獨late EPC、轉染lenti-GFP之EPC以及轉染lenti-VEGF之late EPC以Matrigel培養,經過24小時後觀察其管狀構造形成之能力,我們發現經由lenti-VEGF轉染的細胞,其管長與其他兩組比較,其管狀構造形成之能力的確提升許多。在動物模式方面,我們建立下肢缺血之小鼠模型,在局部肌肉注射轉殖VEGF之late EPC之後,7 天觀察其外表之結果,發現小鼠經手術缺血之下肢之左腳已經回復到與未經手術之右腳相同之外觀。用laser Doppler perfusion imager (LDPI)指數(由缺血下肢與正常下肢之比值來計算)經過評估,顯示lenti-VEGF轉染的late EPC的效果顯現在裸鼠下肢缺血部分血流之再灌注的明顯效果。我們的研究提供了第一個證據,就是以慢病毒攜帶VEGF轉染的late EPC治療下肢缺血之動物模式,在最短時間內可以達到明顯的器官救援。在臨床上應用於病人急性下肢缺血之肢體保存率甚至應用到急性的心肌梗塞上,意味具有顯著的功效。我們希望經由此項研究,能在缺血心血管疾病的治療上,能做為一項有益的參考。
英文摘要 Asahara first isolated the putative endothelial progenitor cells (EPC) from human peripheral blood in 1997. It is believed that EPC home to sites of lesion after ischemia insult, where they differentiate into endothelial cells. Early EPC exhibit a short life span and contribute to neovasculization mainly by secreting angiogenic cytokines, whereas late EPC have a long life span and are known for their robust proliferation and potent vasculogenesis potentials. Angiogenesis has been exploited as one of the strategies in cardiovascular therapy. Vascular endothelial growth factor (VEGF) is an angiogenic factor, which regulates neoangiogenesis to promote vessel growth. Lentiviral vector has several advantages over other vectors, including low immunogenicity, long-term expression, and broad tropism. Combination of EPC with VEGF gene therapy may be a better therapeutic strategy for treating ischemic cardiovascular diseases, such as ischemic legs. The aim of this study is to investigate the therapeutic effect of late EPC lentivirally transduced with human VEGF165 in a mouse ischemic leg model. We constructed lentiviral vectors encoding VEGF (lenti-VEGF) and green fluorescent protein (lenti-GFP) to transduce human cord blood-derived late EPC. The use of the nude mouse femoral artery ligation model enabled us to mimic cardiovascular ischemic diseases. Our results showed that VEGF-transduced EPC were superior to GFP-transduced EPC or EPC alone in enhancing angiogenic effects in vitro and in vivo. Late EPC formed more tubules after cocultured with fibroblasts, a phenomenon which cannot be observed in early EPC. Furthermore, tubule density in Matrigel was significantly higher in late EPC transduced with VEGF than in those transduced with GFP or in late EPC alone. More importantly, in the mouse ischemic leg model, we demonstrated that late EPC transduced with VEGF have the best in vivo limb salvage ability observed 7 days after treatment. Laser Doppler perfusion imager (LDPI) analysis of ischemic limbs indicated that treatment with lenti-VEGF-transduced late EPC could significantly increase blood perfusion. Taken together, our results suggest that VEGF-transduced late EPC may be a promising therapeutic option for ischemic diseases and peripheral arterial diseases. Furthermore, this strategy may have implications for potentially clinical trials in patients with refractory ischemic cardiovascular diseases.
論文目次 中文摘要 I
Abstract Ⅱ
目錄 Ⅲ
圖目錄 Ⅵ
縮寫 Ⅶ
緒論(Introduction):
一、缺血性心臟血管疾病 1
二、內皮幹細胞(Endothelial Progenitor Cells) 2
三、內皮細胞的身份及遺傳表型 4
四、EARLY EPC及LATE EPC 7
五、研究動機與目的 9
材料與方法(Material and Methods)
一、材料
A. 細胞培養液 11
B. 實驗動物 12
C. 麻醉藥品 12
D. 分離單核球細胞之藥品 12
E. 覆蓋培養盤之藥品 12
F. 抗體 12
G. 其他細胞培養用品 13
二、方法
A. 幹細胞分離與細胞培養 13
B. pWPI-vegf 165 質體之構築 14
C. 慢病毒之生產 14
D. 病毒濃縮步驟與效價測定 15
E. 流式細胞儀之確認late EPC 之細胞表面標記 15
F. 轉染的細胞流式細胞儀之GFP螢光表現百分比測定 16
G. 慢病毒之感染內皮幹細胞 16
H. 慢病毒之給予 16
I. 動物大腿骨股動脈之結紮 16
J. 細胞染色 17
K. 免疫組織化學染色分析 17
L. 測細胞上清液中VEGF的蛋白量 17
M. 雷射都普勒(Lasser Doppler Perfusion Image)血流分析 18
N. 測量血管新生 18
O. 免疫螢光染色 18
P. EPC matrigel 管狀構造生成 19

結果(Result)
A. late EPC與early EPC 之培養與特性分析 20
B. EPC經繼代培養擴增後,late EPC細胞表面標記之分析 21
C. 持續觀察繼代之EPC細胞表面標記變化 21
D. 以Matrigel證實late EPC可促進管狀構造之生成 22
E. 表現VEGF慢病毒質體 PWPI–vegf165之構築及慢病毒顆粒之製備 22
F. late EPC轉殖lenti-VEGF以及lenti-GFP後,觀察綠色螢光之表現 23
G. 以流式細胞儀測定late EPC轉殖lenti-VEGF之綠色螢光表現百分比 23
H. 利用Matrigel證實VEGF在管狀構造形成之影響力 23
I. 當與纖維母細胞共同培養時,late EPC自我行成管狀構造之能力 24
J. 用ELISA測定細胞上清液之VEGF含量 24
K. 轉殖VEGF之EPC在裸鼠下肢缺血模式顯示下肢保存之極大優勢 24
L. 以雷射都普勒(laser DOPPLER PERFUSION IMAGER, LDPI)分析實驗動物之血流情形 25
討論(Discussion) 27
參考文獻(References) 31
圖 40

圖目錄
圖一:late EPC與early EPC之型態與細胞表面標記分析 40
圖二:利用流式細胞儀分析late EPC、early EPC和HUVEC的細胞表面標記螢光強度 41
圖三:不同繼代數之late EPC細胞表面標記之分析 42
圖四:以Matrigel證實late EPC可促進管狀構造之生成 43
圖五:表現VEGF慢病毒質體pWPI-vegf165之構築 44
圖六:轉染lenti-VEGF 以及 lenti-GFP 之late EPC,綠色螢光之表現 45
圖七:以流式細胞儀測定轉染lenti-GFP之late EPC之GFP螢光表現量 46
圖八:利用Matrigel證實VEGF在管狀構造形成之影響力 47
圖九:late EPC與MRC-5纖維母細胞共同培養72小時後,自我形成管狀構造之能力 48
圖十:用ELISA測定轉染過lenti-VEGF之 late EPC上清液之VEGF含量 49
圖十一:以雷射都普勒分析(laser Doppler perfusion imager, LDPI)評估下肢缺血之裸鼠經治療後之血流情形 50
圖十二:轉殖VEGF之EPC在裸鼠下肢缺血模式顯示下肢保存之極大優勢 51
圖十三:以雷射都普勒分析((laser Doppler perfusion imager, LDPI)下肢缺血之裸鼠經治療後之血流情形 52
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