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研究生:孫怡惠
研究生(外文):Yi-Hui Sun
論文名稱:以脂肪幹細胞合併內皮前驅細胞促進傷口癒合之研究
論文名稱(外文):To Investigate The Improvement Of Wound Healing Using Adipose Stem Cells Combined Endothelial Progenitors
指導教授:詹益欣詹益欣引用關係
指導教授(外文):Yi-Hsin Chan
口試委員:馬旭
口試委員(外文):Hsu Ma
口試日期:2012-06-04
學位類別:碩士
校院名稱:國防醫學院
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:61
中文關鍵詞:傷口癒合脂肪幹細胞內皮前驅細胞
外文關鍵詞:wound healingadipose stem cellsendothelial progenitor cells
相關次數:
  • 被引用被引用:0
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  • 下載下載:29
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慢性傷口是造成截肢的主要因素,且對於醫療資源的耗費相當龐大,因此如何有效促進傷口癒合是臨床上重要的課題。近年來在慢性傷口以及幹細胞或是前驅細胞的生物學上,有一些新的治療發展,為潰瘍及其他慢性傷口的病人提供了新的希望。內皮前驅細胞(EPC)來自於共同造血前驅細胞,具有高度增殖能力並且能夠形成新血管及修復內皮細胞;人類脂肪幹細胞(hASC)是多能幹細胞,在體外能夠穩定血管的形成。在本篇研究中,我們探討內皮前驅細胞的特性以及人類脂肪幹細胞是否能夠協助內皮前驅細胞形成血管,以作為治療傷口癒合的媒介。我們從脂肪組織分離培養人類脂肪幹細胞,以及從周邊血液分離培養內皮前驅細胞。藉由流式細胞儀、免疫螢光染色及RT-PCR分析細胞特性。結果顯示,人類脂肪幹細胞具有多能細胞發展的能力。而內皮前驅細胞會表現CD34、CD31、CD309、CD133、vWF,並且具有攝取DiI-ac-LDL以及結合UEA-1的功能。利用tube formation assay評估此細胞的功能性,證實內皮前驅細胞在體外具有血管生成的能力。在活體實驗中,我們於裸鼠體側開創傷口後注射內皮前驅細胞、人類脂肪幹細胞及合併注射兩者細胞於傷口周圍,經過20天後,測量傷口面積。利用數位攝影系統及組織化學染色來記錄新血管形成的功能。結果顯示,在合併使用兩者細胞之裸鼠,傷口癒合速度較快且平整,而組織中有較高血管密度,因此合併兩者,具有明顯促進傷口癒合及血管新生的效果。本研究證實,內皮前驅細胞合併人類脂肪幹細胞對於傷口癒合有良好的促進效果,在臨床上具有提供治療慢性傷口的潛能。
Chronic wounds is the major cause of amputation, and consume a lot of medical resources. Therefore, how to effectively promote wound healing is a clinically important issue. Recent advances in our understanding of chronic wound and stem cell or progenitor biology have led to the development of several new treatments that offer renewed hope to patients with ulcers and other chronic wounds. Endothelial progenitor cells (EPCs) are considered to be derived from a common hematopoietic precursor with a high proliferative potential, and have thus been generally thought to contribute to vasculogenesis and repair of endothelial cells. Human adipose stem cells (hASCs) are multipotent cells with pericytic properties that can stabilize vascular assembly in vitro. In this study, we highlighted the characteristics and properties of EPCs, and tested whether hASCs would cooperate with EPCs to co-assemble vessels, which can be promising agents in the therapy of wound healing. We isolated and outgrow human ASCs from fat tissue and EPCs from peripheral blood. Characterization of human ASCs and EPCs was done by flow cytometry, immunofluorescence microscopy, and RT-PCR. The analysis showed that hASCs cultured with multipotent capability and EPC expressed CD34, CD31, CD309, CD133, vWF, and were positive for acetylated LDL uptake and ulex lectin binding and in vitro vasculogenesis capacity. The EPCs were evaluated further for their tube-forming potential using tube formation assay. Then excisional wounds were created on mice and injection the cells of EPCs, hASCs and EPCs combined with hASCs around the wounds for 21 days. In comparison with wound healing, we measured the wound area initial-to-final. Neovascularization was studied using histochemistry staining and digital photography. Mice with wound treated using EPCs combined with hASCs had much more improvement of wound healing and angiogenesis, as they demonstrated by faster healing rate and had more vessel density in wound tissues. These observations suggest that EPCs combined with hASCs could improve wound healing and have a potential for clinical treatment of chronic wounds.
正文目錄 I
圖目錄 IV
中文摘要 V
Abstract VI
第一章 緒論 1
第一節 傷口(wound) 3
壹、 傷口癒合過程 3
貳、 慢性傷口的成因 4
參、 慢性傷口的種類 5
第二節 幹細胞的再生醫學 6
壹、 幹細胞的潛能及來源 6
貳、 胚胎幹細胞 (Embryonic stem cell, ES cells) 7
參、 成體幹細胞 (Adult stem cells) 7
第三節 血管再生治療 9
壹、 內皮前驅細胞 (Endothelial progenitor cells, EPCs) 9
貳、 人類脂肪幹細胞 (Human adipose stem cells, hASCs) 13
第四節 研究動機與目的 15
第二章 材料與方法 17
第一節 實驗材料、耗材及配製方法 17
壹、 Coated-fibronectin 17
貳、 HBSS的配製 17
參、 EGM-2的配製 17
肆、 RBC lysis buffer的配製 17
伍、 10X PBS的配製 18
陸、 5X TBE的配製 18
柒、 2% paraformaldehyde 18
捌、 4% formaldehyde 18
玖、 0.5% BSA 19
壹拾、 1% agarose gel 19
壹拾壹、 細胞的繼代培養及計數 19
第二節 實驗方法 21
壹、 人類週邊血液內皮前驅細胞的分離及培養 21
貳、 螢光染色偵測內皮前驅細胞的表徵 21
參、 流式細胞儀鑑定細胞表面分子之表現 22
肆、 人類周邊血液內皮前驅細胞核醣核酸之萃取 23
伍、 反轉錄酶聚合酶鏈鎖反應(RT-PCR) 24
陸、 體外管腔形成能力分析(Tube formation assay) 25
柒、 動物傷口模式之應用 26
捌、 石蠟組織切片及染色(H&E stain) 27
玖、 冷凍切片及組織包埋 27
壹拾、 免疫組織螢光染色(Immunohistochemistry, IHC) 28
壹拾壹、 細胞染色及追蹤 29
壹拾貳、 統計學分析 30
第三章 結果 31
第一節 人類週邊血液內皮前驅細胞的分離及培養 31
第二節 螢光染色偵測內皮前驅細胞的特徵表現 31
第三節 內皮前驅細胞表面標記的鑑定 32
第四節 內皮前驅細胞相關基因之表現 33
第五節 內皮前驅細胞體外血管新生的功能 33
第六節 傷口外觀癒合的情形 33
第七節 傷口周邊血管分佈的情形 34
第八節 組織切片血管密度分析及統計 35
第九節 傷口組織血管新生因子的表現 35
第十節 內皮前驅細胞參與傷口血管新生的能力 36
第四章 討論 38
參考文獻(References) 42


圖1 自人類週邊血液分離培養之內皮前驅細胞 48
圖2 內皮前驅細胞的特徵表現 49
圖3 內皮前驅細胞表面之標記 50
圖4 內皮前驅細胞基因之表現 51
圖5 內皮前驅細胞體外形成血管的能力 52
圖6 動物傷口癒合之外觀 53
圖7 傷口癒合過程傷口面積之變化 54
圖8 傷口組織血管新生的情形 55
圖9 傷口組織中血管密度的分析 56
圖10 傷口組織中血管新生因子-VEGF的表現 57
圖11 傷口組織中血管新生因子-vWF的表現 58
圖12 傷口組織中血管內皮細胞標記-CD31的表現 59
圖13 細胞標記CM-DiI 60
圖14 追蹤細胞參與傷口組織血管新生的情形 61


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