神經生長因子及GRGDSP/Ln5-P4胜肽修飾膠原蛋白/透明質酸/海藻酸複合水膠自組裝支架培養誘導式多能幹細胞之分化效應

Differentiation of Induced Pluripotent Stem Cells in Nerve Growth Factor-loaded GRGDSP/Ln5-P4 Peptides-Modified Collagen/Hyaluronan/Alginate Self-assembled Hydrogel Scaffold

薛丞宏(Cheng-Hung Hsueh)

指導教授 ： 郭勇志

國立中正大學/工學院/化學工程學系/碩士(2014年)

摘要

本研究採用高含水性的天然高分子膠原蛋白(collgen, COL)、透明質酸(hyaluronan, HA)及海藻酸(alginate, ALG)為基材主要成份，製造適合包覆細胞的三維水膠微環境，並以甲基丙烯酸酐(methacrylic anhydride, MA)改質基材主要三成份，透過光交聯聚合(photocrosslinking)可快速而大量產出可控制形狀的三維水膠微粒(microgel)。水膠微粒於油水兩相系統，藉由兩相界面作用力將重複單元的水膠微粒自組裝(self-assembly)，以底上(bottom-up)方式組成完整的支架。光交聯後的三維水膠微粒環境並不適合細胞生長，因此本研究以胜肽(peptide) GRGDSP及Ln5-P4接枝於高分子鏈網路(polymer network)中，提升細胞在水膠微粒中的包覆率與存活率。誘導式多能幹細胞(induced pluripotent stem cells, iPSCs)具有多能分化能力，透過神經生長因子(never growth factor, NGF)的誘導，讓誘導式多能幹細胞在水膠支架系統中朝向神經分化，形成具神經再生功能的細胞負載(cell-laden)水膠支架。由COLMA/HAMA/ALGMA孔隙度與膨潤度評估比例為1:2:1最佳，並有最佳的線性自組裝排列。支架對細胞包覆率(entrapment efficiency)與存活率(viability)的結果顯示，GRGDSP/Ln5-P4改質水膠可改善iPSCs貼附率約40%及存活率約20%。以anti-SSEA-1及anti--III tubulin進行流式細胞儀分析，Ln5-P4具有神經分化效果，搭配GRGDSP與NGF的作用更能增強分化效果，可達到約90%的神經分化。以anti-SSEA-1、anti--III tubulin及anti-NF-H觀察螢光染色影像，Ln5-P4/GRGDSP改質水膠注入NGF不但能朝像神經分化，並能看到成熟的神經細胞型態。

關鍵字

誘導式多能幹細胞 ； Ln5-P4 ； GRGDSP ； 底上製程 ； 自組裝 ； 光交聯 ； 水膠 ； 細胞負載

並列摘要

In this research, natural polymer collagen (COL), hyaluronan (HA), and alginate (ALG) are chosen as main components of substrate to manufacture a tunable three-dimensional (3D) hydrogel microenvironment for cell entrapment. Three main components modified by methacrylic anhydride (MA) and photocrosslinked to produce numerous 3D microgels immediately. Repeat unit microgels self-assemble by interface force in oilwater two phase systems and compose an integrated scaffold by bottom-up approach. However, 3D microgels are not suitable for cell entrapment and proliferation. Peptides GRGDSP and Ln5-P4 are grafted in polymer network to promote cells entrapped in microgels. Induced pluripotent stem cells (iPSCs) induced by nerve growth factor (NGF) to neuronal differentiation in hydrogel scaffold system, become a nerve regeneration cell-laden. There was an optimal component of hydrogel to swelling ratio, porosity and linear assembly composition, COLMA:HAMA:ALGMA=1:2:1. GRGDSP/Ln5-P4 modified hydrogels improved entrapment efficiency about 40% and viability and 20%. The flow-cytometric analysis against anti-SSEA-1 and anti--III tubulin indicated that Ln5-P4 could induce iPSCs to neuronal differentiation, and Ln5-P4/GRGDSP with NGF could induce neuronal differentiation to almost 90%. Immunochemical staining of iPSCs against anti-SSEA-1, anti--III tubulin, and anti-NF-H, NGF injected Ln5-P4/GRGDSP hydrogels not only induced iPSCs to neuronal differentiation, but became more matural neural cells.

並列關鍵字

induced pluripotent stem cells ； Ln5-P4 ； GRGDSP ； bottom-up ； self-assembly ； photocrosslinking ； hydrogel ； cell-laden

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