臺灣博碩士論文加值系統

內皮前驅細胞能夠修復受傷的血管，促進血管的再生，本實驗分離健康成人之週邊血單核細胞，利用特殊的培養方法，促使貼附性單核細胞分化為類內皮前驅細胞，並鑑定其特徵及功能。自體外培養七天後得到的類內皮前驅細胞，具有攝取 DiI-Ac LDL 以及結合 ULEX-1 的功能，此特徵與前人所報導的內皮前驅細胞相像，並且藉著反轉錄聚合酶連鎖反應以及免疫螢光染色，此細胞也表現內皮細胞之特有基因：VE-cadherin、vWF、eNOS，以及內皮細胞之特有蛋白：vWF。在細胞的功能性分析方面，得知類內皮前驅細胞在體外的細胞間質萃取物 (Matrigel) 上可以促使類似血管的生成；藉由流式細胞儀偵測培養天數不同的細胞表面分子表現：血源性細胞標的：CD105會隨著培養天數大幅降低，另外，早期內皮細胞的內皮血管生長因子接受器1 & 2 (Flt-1 & KDR) 隨著天數表現則逐漸升高，顯示血源性單核細胞轉分化 (trans-differentiation) 的趨勢。此外，在形成類內皮前驅細胞的過程，可以偵測到代表早期細胞的標記基因：Oct-4、Rex-1、Sox-2 的表現逐漸增高，並且細胞端粒酶活性也增強，顯示成人細胞在經由培養之後會表現早期細胞的特性。進一步了解內皮前驅細胞分化過程中分泌了哪些激素，收集其第一天至第四天以及第四天至第七天，類內皮前驅細胞形成過程的培養液，以人類細胞激素蛋白表達微陣列之比對分析，可以得知養成類內皮前驅細胞第四天至第七天，細胞會大量表現：Hemangiogenic factors/Pro-angiogenic factors
(b-FGF、G-CSF and GM-CSF)，Chemokines/Pro-inflammatory factors/Pro-angiogenic factors (GRO/CXCL-2、IL-8/CXCL-8、 I-309/CCL-1、I-TAC/CXCL-11、MCP-1/CCL-2、RANTES/CCL-5) 以及 Cytokine/Pro-inflammatory factors (IL-13) 這些細胞激素，可能經由paracrine或autocrine的機制調控其本身的分化，促使血管的生成及持久性和穩定性，或者扮演其他生理性功能。
　　經由以上的實驗結果證明在體外 (in vitro) 的情況下可以使成人週邊血中的血源系細胞轉分化或去分化成為類內皮前驅細胞，並且可能經由細胞激素的刺激促進血管的再生，進一步將利用動物實驗證實。

This study describes the generation and characterization of human adult peripheral blood mononuclear-derived endothelial progenitor-like cells (EPLCs) as a resource may potentially useful for neo-vasculogenesis.
Peripheral blood mononuclear cell derived EPLCs are characterized by their phonotypical expression of CD34–, CD105+, CD14++, CD31++, CD45+++, and VEGF receptors positive (Flt-1++ and KDR++). These cells up-taking AC-LDL, binding ULEX-1 functions and exhibit tube formation in the Matrigel culture system in resemble to the CD34+, KDR++, AC133+ endothelial progenitor cells (EPCs). EPLCs express endothelial-specific genes such as VE-cadherin, vWF, and eNOS as functioning endothelial cells (ECs). During the course of EPLCs formation culture, increased early genes (Oct-4, Rex-1, and Sox-2) expression and enhanced telomerase activity were detected as their positive cell surface marker CD105 was switched off and at mean time turned on the angiogenic receptors Flt-1, and KDR. In contrast to the previously finding of angiogenic factors G-CSF, VEGF, HGF secreted by the EPCs18, the current study reveals that EPLCs secrete additional hemangiogenic factors/pro-angiogenic factors (b-FGF、GM-CSF), chemokines/pro-inflammatory factors/pro-angiogenic factors (GRO/CXCL-2、IL-8/CXCL-8、 I-309/CCL-1、I-TAC/CXCL-11、MCP-1/CCL-2、RANTES/CCL-5), and cytokine/pro-inflammatory factors (IL-13).
Taken together, the present study demonstrates that EPLCs can be derived from transforming human adult hematopoietic mononuclear cells into functioning endothelial progenitor like cells phenotype via the process of trans-dedifferentiation in vitro that may benefit for the vascular wound healing and regeneration in vivo. Animal model studies of the EPLCs are undergoing.

緒論
　一、細胞治療......................................................01.
　二、胚胎時期血管發育...............................................02.
　三、後生性血管再生.................................................03.
　四、內皮前驅細胞之分離.............................................03.
　五、內皮前驅細胞應用於血管再生治療...................................05.
　六、其他應用於血管再生之幹細胞......................................06.
　七、實驗目的......................................................06.

研究材料與方法
　一、人類週邊血單核細胞的分離及內皮前驅細胞培養........................08.
　二、螢光染色偵測內皮前驅細胞特徵表現.................................08.
　三、免疫螢光染色偵測細胞表面分子表現.................................09.
　四、流式細胞儀鑑定細胞表面分子表現...................................09.
　五、細胞的RNA抽取.................................................10.
　六、反轉錄聚合酶連鎖反應 (reverse transcriptase-polymerase chain reaction, RT-PCR).................................................11.
　七、染色體終端重覆增幅步驟 (telomeric repeat amplification
protocol, TRAP)...................................................11.
　八、體外似管狀構造生成 (tube formation)............................12.
　九、人類細胞激素蛋白表達微陣列 (human cytokine antibody array)......13.

實驗結果與分析
　一、觀察貼附性單核細胞培養後的細胞型態變化............................14.
　二、貼附性單核細胞培養後具有內皮前驅細胞攝取DiI-Ac LDL及結合ULEX-1特徵表現.................................................................14.
　三、貼附性單核細胞培養後表現內皮細胞特殊基因PECAM-1、eNOS、VE-cadherin、vWF及vWF蛋白.......................................................14.
　四、類內皮前驅細胞 (EPLCs) 形成過程中表面分子的變化...................15.
　五、類內皮前驅細胞表現早期細胞標記基因 (Oct-4、Rex-1、Sox2).............................................................16.
　六、類內皮前驅細胞形成過程中細胞端粒酶的活性提高.......................17.
　七、類內皮前驅細胞在體外形成似微管狀構造.............................17.
　八、比較分化為類內皮前驅細胞前後表現的激素............................18.
　九、類內皮前驅細胞表現具有特異性的細胞激素基因 (IL-13、I-309)..........19.

討論...............................................................21.

結論...............................................................28.

表1. 反轉錄聚合酶連鎖反應所使用之引子序列、黏和溫度以及PCR反應次數........29.
表2. 比較內皮前驅細胞之表面分子表現...................................31.
表3. 對照圖九的cytokines的全名、偵測敏感度、差異的比值..................32

圖一、成人週邊血貼附性單核細胞 (adherent PBMNCs) 分化為內皮前驅細胞的型態變化.................................................................33.
圖二、週邊血貼附性單核細胞培養7天後表現內皮前驅細胞的特徵................34.
圖三、Adherent PBMNCs培養後表現內皮細胞之特有基因.....................35.
圖四、Adherent PBMNCs培養後表現內皮細胞之vWF特徵蛋白...................36.
圖五、Adherent PBMNCs分化為類內皮前驅細胞過程中表面分子鑑定.............37.
圖六、內皮前驅細胞表現代表早期細胞的基因...............................39.
圖七、內皮前驅細胞表現端粒酶 (telomerase) 活性........................40.
圖八、內皮前驅細胞在體外生成似管狀構造能力分析..........................41.
圖九、內皮前驅細胞分泌激素分析........................................42.
圖十、內皮前驅細胞表現具有差別的分泌激素基因............................43

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