從臍帶血中建立內皮前驅細胞之探討研究

Development and characterization of endothelial progenitor cells from human cord blood

彭倫浩(Luen-Hau Peng)

指導教授 ： 姚少凌

元智大學/工程學院/化學工程與材料科學學系/碩士(2012年)

https://doi.org/10.6838/YZU.2012.00168

摘要

內皮前驅細胞(endothelial progenitor cell ,EPC)具有分化為成熟內皮細胞的能力，在血管新生 (vasculogenesis)或新生血管形成 (angiogenesis)具有上扮演重要的角色，因此對組織或器官的修復與再生和心血管疾病的治療具有極大的潛力(例如：冠狀動脈梗塞、腦動脈阻塞、周邊動脈堵塞等疾病)，同時再癌症腫瘤的形成上也有著關鍵的影響，所以建立內皮前驅細胞的培養、分離與冷凍保存之系統，須為目前相關領域的重要研究課題。
本實驗利用臍帶血中純化出的單核細胞(mononuclear cells)培養於經由fibronectin前處理的培養系統下在含有胎牛血清及自體血漿的 EGM-2培養基中，成功的建立出內皮前驅細胞，該細胞型態為多角狀以及細胞群集型態為鵝卵石型態，其表面抗原表現出CD31、CD34、CD144、CD105、CD309與vWF在建立內皮前驅細胞後，使用EBM-2培養基添加四種細胞激素( VEGF、b-FGF、IGF-1、EGF )和三種生長因子(Hydrocotision、Heparin、2-phospho-L-Ascrobic acid)進行前培養，進而開發出內皮前驅細胞冷凍保存之培養基，證明細胞經由冷凍後仍具有增殖能力。
此外本實驗根據文獻的探討，將九種細胞激素 (VEGF165、SCGF-α、b-FGF、SCF、IGF-1、FLT-3 ligand、HGF、EGF、IL-8)針對內皮前驅細胞進行測試其促進增殖的能力得知，VEGF165、b-FGF、IGF-1與HGF四種細胞激素對於內皮前驅細胞增殖具有正向幫助，並利用陡昇路徑法，找出其最適化濃度。完成建立本實驗大量量產內皮前驅細胞的增殖系統。
在建立最適化培養基之後，進一步再檢測內皮前驅細胞的特性，如典型內皮表面抗原分析、Ac-LDL吞噬能力 (take-up)形成網絡血管能力(Matrigel angiogenesis assay)，經由以上實驗的證明，經由最適化濃度培養基培養十二天後，增殖後的內皮前驅細胞仍具有以上功能性。
最後，本研究建立之內皮前驅細胞體外分離、鑑定、培養與冷凍保存的系統，可大量產生具有功能性的內皮前驅細胞，以供後續基礎研究與臨床應用之所需。

關鍵字

內皮前驅細胞 ； 臍帶血 ； 血管新生

並列摘要

In human, angiogenesis is not only important for physiology and development, but also correlation with most cardiovascular diseases such as myocardial infraction. Recently, many reports further demonstrated that angiogenesis related closely with tumor development. Angiogenesis and vasculogenesis are regulated by the proliferation and differentiation of endothelial progenitor cells (EPCs).
In this study, we isolated mononuclear cells (MNCs) from human umbilical cord blood and cultured MNCs in the EGM-2 medium. According to the results, several cytokines, especially VEGF165, b-FGF, HGF and IGF-1, have been identified as essential factors to proliferation.
In addition, throughout culture cells preserved their functional capacity that was demonstrated by high uptake of DiI fluorescently conjugated- acetylated-low density lipoprotein (DiI-Ac-LDL), and in vitro tube formation. Flow cytometry demonstrated that EPCs expression of endothelial progenitor markers was not affected by long term in vitro culturing, such as CD31, CD34 , CD105, CD144, CD309 and vWF .
Our results showed that EPCs derived from human umbilical cord blood sample exhibited both characteristics and functions of EPCs. Therefore, we believed EPCs expansion medium would generate large amounts of functional EPCs.

並列關鍵字

endothelial progenitor cells ； human umbilical cord blood ； angiogenesis

參考文獻

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