2017-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/644631摘要：糖尿病患的困難傷口修復已成為一個臨床上的重大課題，且對病患以及整體社會造成經濟上的巨大負擔。迄今已有許多治療糖尿病患傷口的方法，然而這些方法仍存在治療上的限制。因此，研發新的治療方式，如幹細胞療法，被認為是解決此問題的良方。脂肪幹細胞的來源豐富且容易取得，是極具潛力的幹細胞治療工具。然而，之前的研究發現糖尿病病患自體來源的脂肪幹細胞因為血管新生能力及再生潛能受損，故對於糖尿病病患傷口治療效果有限。此外，從需要幹細胞治療的糖尿病病患身上收集脂肪幹細胞是高花費且較無效率的作法。相對上，若能解決免疫排斥反應的問題，取健康捐贈者來源的脂肪幹細胞進行異體移植是個更可行的治療方法。脂肪幹細胞除了具有多能分化能力，其免疫調節特性亦是近來備受矚目的焦點。當臨床上出現如異體細胞移植等啟動免疫反應的情形時，脂肪幹細胞便適合應用於扮演的免疫調節角色。之前我們觀察到利用幾丁聚醣膜培養脂肪幹細胞形成三維球體，會提升其幹細胞特性及分化能力，另外有其他研究指出三維球體培養可以促進中胚層幹細胞的免疫調節能力。因此在本計畫中，我們將利用微流道技術，以幾丁聚醣/明膠混合作為原料，製作孔徑一致且互相連通的三維多孔支架。此支架可使脂肪幹細胞於孔洞中自動形成三維球體，同時增加脂肪幹細胞球體異體移植至糖尿病病患傷口的便利性。我們假設支架中的脂肪幹細胞球體會增強其免疫調節能力，使異體移植的脂肪幹細胞球體可避開免疫排斥反應，並且促進糖尿病病患之傷口修復。我們已初步製作出孔徑一致且互相連通的三維多孔明膠支架，不僅發現支架中的脂肪幹細胞有聚集的趨勢，且脂肪幹細胞表現出來的特性與平面培養時的特性不同。因此，在本計畫的第一年，我們預計將幾丁聚醣加入此三維多孔支架中，目的在於促進脂肪幹細胞在支架孔洞中形成細胞球體，並且觀察支架中脂肪幹細胞球體表現的細胞特徵。計畫的第二年，我們將深入探討支架中脂肪幹細胞球體的幹細胞特性、血管新生能力以及免疫調節能力。而在計畫的第三年，我們將利用糖尿病小鼠傷口癒合模式進行測試，將三維多孔支架及其中形成的脂肪幹細胞球體植入小鼠傷口中觀察傷口修復的情形。本計畫的研究目的在於利用此新穎的三維培養技術，進而增強脂肪幹細胞的再生能力及免疫調節能力。當完成此研究計畫時，我們期許能研發出使脂肪幹細胞自主形成細胞球體的三維多孔幾丁聚醣/明膠支架，並將此支架運用在脂肪幹細胞異體移植治療上，而對糖尿病病患的傷口修復有所助益。<br> Abstract: Poor wound healing in diabetic patients has become a major clinical challenge with enormoussocioeconomic burden. Many treatment options are available nowadays for diabetic wounds, but they allhave certain limitations. Therefore, it is important to develop novel treatments, such as cell therapy, to tacklethis problem. The abundance and easy accessibility of adipose-derived stem cell (ASC) have made it an idealcandidate for stem cell therapies. However, using autologous ASCs for diabetic wounds exhibits a majorconcern that diabetic ASCs are impaired in their angiogenic and regenerative potentials. Moreover,harvesting ASCs from each diabetic patient who requires cell therapy renders the procedure expensive andinefficient. Allotransplantation of ASCs from healthy non-diabetic donors appears to be a feasiblealternative approach, but the issue of immune rejection has to be addressed.Besides their potential to undergo multilineage differentiation, the immunomodulatory properties ofASCs have recently drawn much attention. Their immunoregulatory role can be applied in several clinicalconditions in which an immune response is present, such as allogenic cell transplantation. Moreover, wepreviously observed enhanced stemness and differentiation capabilities in three-dimensional (3D) spheroidculture system of ASCs on chitosan films, and other studies suggested that spheroid formation also enhancethe immunomodulatory capabilities of mesenchymal stem cells. In this proposal, we plan to fabricate a 3Dchitosan/gelatin scaffold with identical interconnected pores using microfluidic technique. The scaffoldcan support spontaneous ASC spheroid formation within the pores, thus facilitating the allotransplantation ofASC spheroids into diabetic wounds. We hypothesize that the ASC spheroids exhibit superiorimmunomodulatory properties, so the transplanted allogenic ASCs can circumvent immune rejectionand promote diabetic wound healing.In our preliminary study, we have fabricated a 3D porous gelatin scaffold with identical interconnectedpores. ASCs cultured in the scaffold showed a tendency toward aggregation and exhibited distinct cellularcharacteristics compared to monolayer culture. Therefore, in the first year of this proposal, we will addchitosan into the 3D scaffold to foster spheroid formation of ASCs within the pores, and we will characterizethe seeded ASCs. In the second year, we will further explore the stemness, angiogenic and immnomodulatoryproperties of the ASC spheroids in the 3D scaffolds. In the third year, we will utilize a wound model indiabetic mice to test the effectiveness of scaffold-facilitated human ASC spheroid transfer to promote woundhealing. The proposed work is designed to explore and utilize the inherent regenerative andimmunomodulatory capabilities of ASCs by a novel 3D culture method. At the completion of this project, weexpect to develop a 3D porous scaffold that enables spontaneous ASC spheroid formation, thereby canbe applied in ASC allotransplantation to promote diabetic wound healing.糖尿病傷口脂肪幹細胞細胞球體免疫調節異體細胞移植diabetic woundadipose-derived stem cellcell spheroidimmunomodulationcell allotransplantation