奈米纖維基材於神經幹細胞分化之表觀遺傳調控機制之研究

The Effects of Electrospun Nanofibers on Epigenetic Regulations during Neural Stem Cell Differentiation

麥卡多(Augustus T. Mercado)

指導教授 ： 葉瑞銘 ； 陳中庸

中原大學/理學院/化學研究所/博士(2017年)

https://doi.org/10.6840/cycu201700235

摘要

針對不同的奈米基材評估表觀遺傳調節其參與之神經幹細胞(NSC) 的分化與發育是目前生物醫學應用所需要的。在此研究中使用兩種電仿絲 奈米纖維(electrospun nanofibers)，分別是聚鄰甲氧基苯胺(poly(o- methoxyaniline, POMA)以及四乙基原矽酸鹽奈米纖維(tetraethyl- orthosilicate nanofiber, SNF-AP)。利用在 POMA 上分化的神經幹細 胞進行聚合酶連鎖反應的選擇性抑制雜交(PCR Selective Suppression Hybridization, PSSH)以提供其 DNA 全基因體甲基化圖譜，然後利用 BLAST 對 PSSH 的篩選基因進行測序和鑑定，再使用 Gene Set Toolkit (Gestalt)、Database for Annotation、Visualization and Integrated Discovery (DAVID) 以及 Pathway Interaction Database (PID) 整理標記其表觀遺傳調節 之基因的功能圖譜。在幹細胞於奈米基材分化後，我們挑選十ㄧ個被甲基 化的功能基因。此外，透過 miRNA 微陣列分析(microarray analysis)分 化前後 NSC 的 miRNA 圖譜發現，與平面基質(flat substrate)相比， POMA 的奈米纖維能誘導更多 miRNA 的表達。相反地，通過微陣列分析 鑑定了在 SNF-AP 生長的 NSC，miRNA 與 mRNA 有不同的表達。利用全 生理路徑分析(Ingenuity Pathway Analysis, IPA)NSCs 發育的分化基因與 候選 miRNA 發現，SNF-AP 刺激負責神經元的增殖、發育和生長、細胞分 化和增生、軸突形成、軸突的生長、微管動力學、細胞突起的形成以及 NSC 分化期間的長期增強基因的表達;然而，相對於聚-D-賴氨酸(poly- d-lysine, PDL)有抑制神經發生、微管動力學、細胞的增殖與分化以及刺 激細胞凋亡等現象。SNF-AP 還促進更多 let-7 miRNA 的表達。總體而言， NSC 分化成神經組織在 POMA 和 SNF-AP 是生物工程中有用的基材。

關鍵字

奈米纖維 ； 神經幹細胞 ； 分化 ； 表觀遺傳學

並列摘要

An evaluation of the genetic regulations involved in neural stem cell (NSC) differentiation towards different artificial environments is needed for its biomedical applications. Two electrospun nanofibers, poly(o-methoxyaniline) (POMA) and tetraethyl-orthosilicate nanofiber (SNF-AP), were used in this study. NSCs differentiated on POMA underwent PCR Selective Suppression Hybridization (PSSH) to provide its DNA methylation profile. DNA fragments were sequenced and distinguished through BLAST. Then, identified genes were annotated using Gene Set Toolkit (Gestalt), Database for Annotation, Visualization and Integrated Discovery (DAVID) and Pathway Interaction Database (PID). Eleven genes corresponding to stem cell functions were methylated after differentiation on the nanomaterial. Moreover, the miRNA profiles of NSC before and after differentiation were assessed by miRNA microarray. POMA nanofibers induced the expression of more miRNAs compared to the flat substrate. Conversely, differentially expressed mRNAs and miRNAs of NSCs grown on SNF-AP were identified through microarray analysis. The genes and miRNA targets responsible for the differentiation fate of NSCs and neuron development process were determined using Ingenuity Pathway Analysis (IPA). SNF-AP stimulated the expression of genes responsible for proliferation, development, and outgrowth of neurons, differentiation and generation of cells, neuritogenesis, outgrowth of neurites, microtubule dynamics, formation of cellular protrusions, and long-term potentiation during NSC differentiation. However, poly-d-lysine (PDL) inhibited neuritogenesis, microtubule dynamics, and proliferation and differentiation of cells and activated apoptosis. SNF-AP also promoted the expression of more let-7 miRNAs. Overall, POMA and SNF-AP are useful scaffolds for NSC differentiation in the development of neural tissue engineering. These findings can be applied for the enhancement of vitro NSC differentiation potential for preclinical studies and future clinical purposes.

並列關鍵字

nanofiber ； neural stem cells ； differentiation ； epigenetics

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