2015-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/644617摘要：來源豐富且易取得的脂肪幹細胞（adipose-derived stem cell)是一理想且具潛力的幹細 胞治療工具。除了具有多能分化潛力外，脂肪幹細胞的免疫調節能力近來備受重視，因其有多 方面之臨床應用，例如產生異常免疫反應的自體免疫疾病，或在進行器官移植時予以免疫抑制 等。我們已經成功建立數個三維培養系統，且發現利用此培養系統可以提升脂肪幹細胞的再生 功能與分化能力。在這次計晝中，我們將利用體外及體内試驗進一步探討脂肪幹細胞的免疫調 節功能，並預期利用三維培養系統可以提升脂肪幹細胞的免疫調節能力。我們將製備下列三維培養系統，並研究其中脂肪幹細胞的免疫調節特性：多孔明膠鷹架 (porous gelatin scaffold)、褐藻膠微粒（alginate microsphere)、細胞球體（cell spheroid)和細胞薄 片（cell sheet)。我們的前期實驗顯示，細胞球體或細胞薄片培養方式可以使脂肪幹細胞減少分 泌促發炎細胞激素interleukin-8。此外，我們重新檢視先前動物實驗的傷口切片，發現以脂肪 幹細胞薄片治療的傷口有明顯較少巨噬細胞聚集的現象，暗示脂肪幹細胞在薄片三維結構中具 較高的免疫調節能力。本計晝中，我們將深入地探討三維培養系統中的脂肪幹細胞，在受到發炎反應刺激時所分 泌的免疫調節因子，例如：prostaglandin E2 (PGE2)、indoleamine 2,3-dioxygenase (IDO)、 transforming growth factor-01 (TGF-01)及interleukin-6。以外，我們將收集人類周邊血液單核細 胞（peripheral blood mononuclear cells)並從中分離出不同的免疫細胞，再將這些不同的免疫細胞分別與三維培養的脂肪幹細胞進行共同培養，以此探討脂肪幹細胞免疫調節的能力。最後， 我們將在糖尿病動物傷口癒合模式中進行三維培養的脂肪幹細胞異體移植，藉由糖尿病傷口之 發炎環境及細胞異體移植可能引起之免疫排斥，我們可深入探討脂肪幹細胞其在免疫調節上的潛力。本計晝的目的是透過新穎的三維培養方法，我們探索如何加強脂肪幹細胞既有的免疫調節 能力。當完成此次計晝時，我們期許能夠建立評估脂肪幹細胞的免疫調節能力之標準實驗程 序。我們也期許能夠開發最適當的三維培養方法，可以用來促進脂肪幹細胞的免疫調節及抗發 炎反應的潛力。藉由三維培養技術之功能促進作用，我們相信脂肪幹細胞可廣泛應用於多種免 疫調節的臨床治療。<br> Abstract: The abundance and easy accessibility of adipose-derived stem cell (ASC) have made it an ideal candidate for stem cell therapies. Besides their potential to undergo multilineage differentiation, the immunomodulatory properties of ASCs have recently drawn much attention. Their immunoregulatory role can be applied in several clinical conditions in which an immune response is present, such as autoimmune diseases; or feared, such as organ or cell transplantation. We have successfully fabricated several three-dimensional (3D) culture systems and observed enhanced stemness and differentiation capabilities under such conditions. In this proposal we plan to investigate the role of ASCs in immunomodulation in vitro and in vivo, and test our central hypothesis that the immunomodulatory properties of ASCs may be enhanced in 3D culture environment.Specifically, we plan to examine the immunomodulatory properties of ASCs in the following 3D culture conditions: porous gelatin scaffold, alginate microsphere, cell spheroid and cell sheet. In our preliminary study, we found decreased levels of inflammatory cytokines, such as interleukin-8, in conditioned medium of ASC spheroids or sheets. Moreover, we re-examined the wound sections in previous animal studies and found significantly less macrophages present in the cutaneous wounds treated with ASC sheets.Based on our encouraging preliminary data, we propose to further investigate the secretion of immunomodulatory factors (e.g., prostaglandin E2, indoleamine 2,3-dioxygenase, transforming growth factor-pi and interleukin-6) in 3D culture systems of ASCs under inflammatory stimuli. Moreover, we will harvest human peripheral blood mononuclear cells and further isolate different immune cells for co-culture studies with 3D-cultured ASCs to elucidate their immunosuppressive functions. Finally, we will examine the potential of 3D-cultured ASCs in regulating the immune system in the context of cell xenotransplantation for diabetic wound healing in an animal model.The proposed work is designed to explore and utilize the inherent immunomodulatory capabilities of ASCs by novel 3D culture methods. At the completion of this project, we expect to establish feasible in vitro and in vivo protocols to evaluate ASC immunomodulatory properties.We also expect to develop an optimal 3D culture approach for potential ASC application with purposes of immunosuppression or anti-inflammation. By applying 3D culture technology, we believe that ASCs exhibit great potentials in a broad range of immunomodulatory therapeutics.脂肪幹細胞三維培養系統免疫調節抗發炎細胞移植adipose-derived stem cell3D culture systemimmunomodulationanti-inflammationcell transplantation