奈米基材促進神經幹細胞生長分化之DNA甲基化模式研究

神經幹細胞分化在許多的應用中提供了廣泛且多樣的選擇，例如細胞治療或是基因治療，因此要全面的了解神經幹細胞內部的機制，我們必須由基因的層次上開始探討。表觀遺傳學涵蓋了很多方面，例如在不改變基因序列的情況下進而改變基因的表現或是調控神經幹細胞在分化成各種不同類型細胞時大部分的機轉，因此在神經幹細胞的機轉中闡明表觀遺傳調控的規則是很重要的，這個實驗的目的是為要找出在神經幹細胞於不同的基材中分化時，有哪些相關的機制涉及表觀遺傳的修飾調控；基因的表現與不表現分別在未分化和分化的細胞中將被經由篩選揭示出與分化相關的功能與路徑。為了這個目的，從培養在POMA的奈米材料上的細胞和神經幹細胞中純化出去氧核醣核酸，並且經由亞硫酸氫鈉處理、USER酵素的作用反應、生物素的標記、切口的接合、使用生物素來分離甲基化及未甲基化的去氧核醣核酸，再經adapter接合反應、PCR選擇性抑制雜交、TA-選殖以及選殖插入片段的分析和最後的基因序列定序。透過 Gene Set Toolkit (Gestalt) database 找出 12 個與分化相關的基因，其中有 6 個基因已被文獻報導過 (F2R, KIF5C, NCAM2, RB1, FZR1, and ZEB1)，有4個基因被視為候選基因 (TIAM1, DCC, SLCO4C1, TCL1A)，而有 2 個基因被預測可能成為候選基因 (USH1C, ZMIZ1)。另一方面來說，經由 DAVID資料庫有7個基因被鑑別為與分化相關；其中有4個基因已被證實 (HCN1, DMD, KIF5C, NCAM2)，1個為候選基因 (DCC)，1個基因被預測與分化有關 (USH1C)，以及有1個基因尚未被證實涉及分化反應 (CNTNAP4, contactin associated protein-like 4)。只有 KIF5C、 NCAM2、 DCC、 USH1C 和 DMD 基因是同時經由兩個生物資訊工具證實的。利用 Pathway Interaction Database (PID)，將上述基因之間的相關性於生理路徑中呈現出來。Netrin-mediated 訊息傳遞路徑被懷疑可能參與神經幹細胞的分化,因為經由Gestalt 和DAVID證實DCC基因是包含在這兩個路徑當中。基因甲基化的辨別確認了培養在POMA基質並用dbcAMP處理的神經幹細胞已成功分化，某些神經特異基因像是 KIF5C 和 ELMO1經由DNA甲基化的方式被關閉造成第七天時，細胞型態轉成神經膠細胞，證明了星狀細胞分化經這些基因調控的可能性。

關鍵字

DNA 甲基化；神經幹細胞分化

並列摘要

The differentiation of neural stem cells offers a wide variety of options in many applications, such as in cellular therapy and gene therapy; hence to fully understand the inner machinery of neural stem cells, we have to begin at the genetic level. Epigenetics, which encompasses changes in gene expressions without imposing alterations in the genetic sequence, governs most of the mechanisms that lead to neural stem cell differentiation into different types of cells. Therefore it is important to elucidate its roles in the neural stem cell machinery. The objective of this study is to identify epigenetic modifications to discover related mechanisms involved in neural stem cell differentiation. Genes expressed and unexpressed in both undifferentiated and differentiated cells will be screened to reveal relevant pathways and functions associated with differentiation. For this purpose, genomic DNA extracted from cells cultured on different biomaterials and NSCs were subjected to a series of steps beginning with Bisulfite treatment, USER enzyme reaction, biotinylation, ligation of nicks, separation of unmethylated and methylated DNA using Streptavidin, Mung Bean Nuclease reaction, adapter ligation, PCR seletive suppression hybridization, TA cloning, clone insert analysis, and finally gene sequencing. Twelve differentiation genes were identified by Gene Set Toolkit (Gestalt) database: 6 confirmed by literuature reports (F2R, KIF5C, NCAM2, RB1, FZR1, and ZEB1), 4 considered as candidate (TIAM1, DCC, SLCO4C1, TCL1A), and 2 predicted to be candidates (USH1C, ZMIZ1). On the other hand, 7 genes were identified by DAVID to be differentiation-related; 4 were confirmed genes (HCN1, DMD, KIF5C, NCAM2), 1 candidate gene (DCC), 1 predicted (USH1C), and one whose involvement in differentiation is not yet fully explored (CNTNAP4, contactin associated protein-like 4). Only KIF5C, NCAM2, DCC, USH1C, and DMD were validated by both Bioinformatics tools. Based on the Pathway Interaction Database (PID), some of these genes are indirectly related by pathways. Of the identified pathways, netrin-mediated signaling and pathways in cancer may be associated with neural stem cell differentiation as DCC, which was confirmed by Gestalt and DAVID, is involved in them. Identification of methylated genes confirmed the successful differentiation of NSCs on POMA substrate with dbcAMP treatment, while silencing of neuron-specific genes such as KIF5C and ELMO1 through methylation supported the possibility of astrocyte differentiation, as evident from the morphology of cells at Day 7, which showed early glial-like features.

並列關鍵字

DNA methylation ； Neural stem cell differentiation

參考文獻

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奈米基材促進神經幹細胞生長分化之DNA甲基化模式研究

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