奈米基材對神經幹細胞貼附分化之表觀遺傳學研究

Epigenetics Involved in the Adhesion and Differentiation of Neural Stem Cell on Fibrous Matrices

劉尚豪(shang-Hao Liu)

指導教授 ： 陳中庸

中原大學/工學院/生物科技研究所/碩士(2014年)

https://doi.org/10.6840/cycu201400652

摘要

近年來，神經幹細胞治療在組織工程中有著重要的意義，研究幹細胞分化調控對臨床醫療神經修復是當重要研究議題。表觀遺傳的變化被證實主要在負責胚胎的細胞分化與胎兒的發育之基因調控。了解表觀遺傳在神經幹細胞分化上的機轉及各種調控可使神經幹細胞之醫療用途更廣。此外，對奈米材料與神經幹細胞分化之相互關係進行外遺傳機制分析，除確保材料對神經幹細胞基因結構的安全性，也進一步釐清材料影響神經幹細胞分化之關係。因此對神經幹細胞分化在電紡納米纖維基材上功能的遺傳和表觀遺傳的效果進行了分析。這項研究的目的是評估參神經幹細胞在人工基材分化的表觀遺傳修飾，找到神經幹細胞分化和人工基材之間的相關途徑，在分化過程中哪些基因會因環境的關係而發生甲基化。將神經幹細胞培養在基材上時，可以看見哪些甲基化的神經幹細胞基因會造成增殖，生長和分化，從不同合成的基材培養分化的神經幹細胞和未分化的神經幹細胞中分離出脱氧核糖核酸(DNA)及核糖核酸(RNA)。我們從奈米材料AP3M中找到8個候選基因，從奈米材料POMA中找到5個候選基因，從生物資訊方法IPA所找到的13個候選基因中ERBB2, KIF5C, NCAM2, ELMO1, IGF1, NDEL1, NGF和CDH2這8個基因的表現量會上升。利用亞硫酸氫鹽測序法檢測(BSP)和即時聚合酶鏈反應(RT-PCR)來確認這些候選基因的DNA甲基化CpG島及基因的表現量。結果顯示當神經幹細胞分化於奈米纖維基材上有6個基因有發生甲基化的情形，評估奈米纖維材料在表觀遺傳機制於神經幹細胞在分化中的變化有助於對於神經幹細胞的臨床醫學修復上的應用。

關鍵字

奈米基材 ； 神經幹細胞 ； 表觀遺傳學

並列摘要

In recent years, neural stem cells (NSCs) therapy holds great importance in tissue engineering. Understanding the control mechanisms of stem cell differentiation for clinical neural therapy is an important research issue. Also, epigenetic regulations of genes were critical in embryonic cell differentiation and fetal development. Therefore, knowledge on the epigenetic mechanisms for NSCs differentiation can provide various and broad applications for regenerative medicine. In addition the effect of a material on the differentiation of NSCs in relation to its epigenetic mechanisms will ensure its safety. The aims of this study are to investigate the effect of the electrospun nanofibrous substrates on the functional genetics and epigenetic profiles of NSC differentiation and identify differentially methylated genes, and to find relevant pathways involved in the NSC differentiation on artificial micro environments. The genomic DNA and RNA are extracted from differentiated and undifferentiated NSCs to confirm the expression of the chosen candidate genes. The genes responsible for the proliferation, growth, and differentiation fate of NSCs are evaluated before and after its differentiation on amino-propyl silica (AP3M) and poly-o-methoxyaniline (POMA). We found eight candidate genes from AP3M and five candidate genes from POMA involved in the NSCs differentiation on three different nanofibrous substrate. In Ingenuity Pathways Analysis (IPA), the gene expressions of eight genes from thirteen candidate genes increased, such as ERBB2, KIF5C, NCAM2, ELMO1, IGF1, NDEL1, NGF and CDH2. To conform the microarray analysis, Bisulfite sequencing PCR (BSP) and real-time PCR (RT-PCR) were verified the DNA methylation of CpG islands and gene expression of these candidate genes. The data demonstrated six genes with methylation changed when NSCs differentiation on nanomaterial. An assessment on the changes of epigenetic mechanism during NSC differentiation in an inorganic nanomaterial will be useful for the medical application of NSCs.

並列關鍵字

Fibrous Matrices ； Neural Stem Cell ； Epigenetics

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