表面修飾activin A之明膠-聚乳酸聚甘醇酸奈米粒子支架與LY294002及視黃酸共培養誘導式多能幹細胞之分化效應

Differentiation of induced pluripotent stem cells in activin A-grafted gelatin-PLGA NP scaffold co-culture with LY294002 and retinoic acid

劉育銓(Yu-Chuan Liu)

指導教授 ： 郭勇志

國立中正大學/工學院/化學工程學系/碩士(2015年)

摘要

本研究以明膠(gelatin)、聚乳酸聚甘脂酸奈米粒子(poly (lactide-co-glycolide) nanoparticle, PLGA NP)為支架主體，以不同重量比例混合後，透過冷凍乾燥法(freeze-drying)進行製備，討論改變材料組成比例對支架物理性質及生化特性，同時表面以化學交聯方式1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)與N-hydroxysuccinimide (NHS)將activin A交聯在支架表面上，並與LY294002作用對誘導式多能幹細胞(induced pluripotent stem cells, iPSCs)進行體外培養，達到朝內胚層細胞分化結果，再以視黃酸(retinoic acid, RA)作用朝胰島分化效應。以不同PLGA NP比例調控支架孔隙度、含水率，隨著gelatin濃度與PLGA NP比例增加，孔隙度與膨潤度下降，利用電子顯微鏡觀察PLGA NP與gelatin-PLGA NP支架，可觀察PLGA NP為圓球狀，且支架表面上的粒子隨著PLGA NP比例增加而增加，增加支架表面粗糙度。分別對誘導式多能幹細胞進行體外培養。由孔隙度、含水率、接枝率評估明膠-聚乳酸聚甘脂酸奈米粒子支架中gelatin濃度為5%，PLGA NP比例為6.7%時最佳。支架對細胞貼附效率(adhesion efficiency)的結果顯示，當改質的activin A濃度為100 ng/ml可使貼附效率提升約3.9%。以anti-SSEA-1、anti-SOX17做免疫螢光染色，結果表明activin A改質的支架可顯著提升iPSCs分化成內胚層，與LY294002作用之activin A改質的支架可顯著表現SOX17，以anti-SOX17、anti-PDX1做免疫螢光染色，結果表明RA可顯著提升內胚層細胞朝胰島分化。另外以流式細胞儀對anti-SSEA-1和anti- SOX17分析，結果顯示activin A濃度為100 ng/ml時，內胚層比例提升了59.36%，而以LY294002與activin A改質的支架作用則可發現內胚層細胞發展得更成熟且SSEA-1比率也降低，以anti-SOX17和anti- PDX1分析，結果顯示RA濃度為2 μM時，PDX1+比例達到80.63%。

關鍵字

LY294002 ； activin A ； 聚乳酸聚甘醇酸奈米粒子 ； 誘導式多能幹細胞 ； 視黃酸

並列摘要

In this research, the surfaces of gelatin-(poly (lactide-co-glycolide) nanoparticle, PLGA NP) scaffolds were modified with activin A treated with LY294002 and retinoic acid (RA) guides the differentiation of induced pluripotent stem cells (iPSCs) toward endodermic and pancreatic cells. The gelatin-PLGA NP scaffolds were fabricated by freeze-drying method. An increase in the weight percentages of PLGA NP decreased the porosity and swelling ratio of gelatin-PLGA NP scaffolds. The images of FE-SEM revealed that the morphology of PLGA NPs is spherical and the surface of gelatin-PLGA NP scaffolds has more PLGA NPs as an increase in the weight percentages of PLGA NPs. In this research, we manufactured three-dimensionally gelatin- PLGA NP scaffolds by freeze-drying method. The pore surfaces were grafted with activin A by EDC and NHS conjugating agent. When the weight percentages of PLGA NPs in gelatin-PLGA NP scaffolds of 6.7% is the best for scaffold composition. Adhesion efficiency of iPSCs in activin A-grafted (100 ng/ml) scaffold increased 3.9% compared to unmodified scaffold. Immunofluorescent staining indicated activin A could guide iPSCs to differentiate toward endodermic cells. RA could guide endodermic cells to differentiate toward pancreatic cells. The flow cytometry analysis indicated that the SOX17-positive cells were proportional to the amounts of activin A. The percentage of SOX17 increased 59.36% of activin A-grafted (100 ng/ml) scaffolds compared to unmodified scaffold. Activin A-grafted scaffolds treated with LY294002 guided iPSCs differentiate toward endodermic cells significantly compared to activin A -grafted scaffolds. The flow cytometry analysis indicated that the percentages of PDX1 increased 80.63% of activin A-grafted (100 ng/ml) scaffolds treated with LY294002 (10 μM) and RA (2 μM) compared to scaffolds treated without RA. The proper control over the physical and biomedical properties of the scaffolds can guide iPSCs toward endodermic and pancreatic differentiations for diabetes treatment.

並列關鍵字

LY294002 ； activin A ； poly (lactide-co-glycolide) nanoparticles ； induced pluripotent stem cells ； retinoic acid

參考文獻

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