| Summary: | 碩士 === 中原大學 === 生物科技研究所 === 101 === A detailed collaboration of proteomic, genomic, and epigenomic analyses of neural stem cells (NSCs) in synthetic microenvironments will help us resolve some of the numerous questions in regards to regenerative medicine and therapeutic treatment of human diseases. Hence, to fully understand its properties, NSC differentiation on electrospun nanofibrous substrates and the effect on both their functional genetic and epigenetic profiles were analyzed. The objective of this study is to identify the changes in the epigenetic mechanisms involved in the NSC differentiation in an artificial, inorganic matrix. Differentially methylated genes and differentially expressed miRNA targets of NSC differentiated on aminopropyl silica nanofiber (AP3M) were identified to determine its effect in this cellular process. AP3M is a chemically-modified, electrospun nanofiber made from tetraethyl-orthosilicate. The genes responsible for the proliferation, growth, and differentiation fate of NSCs could be miRNA targets and/or differentially methylated when NSCs are cultured in such substrates. To specifically identify these genes, the genomic DNA extracted from cells cultured and differentiated on AP3M, and undifferentiated NSCs were subjected to a whole epigenomic screening method designated PCR Selective Suppression Hybridization (PSSH). However, we were not able to identify any gene from PSSH. Through the microarray analysis of the differentially expressed miRNAs, predicted gene targets during differentiation were identified with the aid of Ingenuity Pathway Analysis (IPA). Moreover, IPA clustered the target genes according to its function and role in the nervous system development. There were 5 notable gene clusters; neuritogenesis, morphogenesis of neurites, differentiation of neurons, cell viability of neurons, and growth of neurites. On the basis of the target genes classified by IPA, AP3M induced the synaptic formation of differentiated and mature neurons after five days of culture with dbcAMP. Genes involved in this process are CDH2, SNCA, RELN and NOTCH3, which were predicted to be differentially upregulated gene targets only for AP3M only. Furthermore, DPYSL2 or CRMP2 was predicted to be downregulated in AP3M but upregulated in PDL, which just induced early NSC differentiation. Also, GDNF is upregulated both in PDL and AP3M. This gene has a significant role in promoting the survival and differentiation of neurons. However, there are important genes (NGF, NTRK2, and NDEL1) for the neuron development and differentiation that are downregulated in AP3M. The gene expression of all the genes mentioned needs to be confirmed since these were just predicted targets from the miRNA profile. Overall, AP3M is a worthy candidate as a scaffold for neural tissue engineering through NSC differentiation.
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