運用高通量基因微矩陣列方法解析由嗜鉻細胞分化成神經細胞之全基因體的調控;Using high-throughput arrays to identify genome-wide regulatory elements and gene transcription in the neuron-differentiation of PC12 cell line

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題名:	運用高通量基因微矩陣列方法解析由嗜鉻細胞分化成神經細胞之全基因體的調控;Using high-throughput arrays to identify genome-wide regulatory elements and gene transcription in the neuron-differentiation of PC12 cell line
作者:	邱佩洵;Pei-hsun chiu
貢獻者:	系統生物與生物資訊研究所
關鍵詞:	細胞神經生長因子微陣列;PC12 NGF microarray
日期:	2009-06-29
上傳時間:	2009-09-22 10:22:49 (UTC+8)
出版者:	國立中央大學圖書館
摘要:	基因調控在細胞生長及分化等生物過程中扮演非常重要的角色，而影響基因調控之重要因素之一主要來自於基因本身與蛋白質相互作用的行為以及被調控基因產生之蛋白質對其他基因的回饋性調控。要了解複雜的基因調控-表達-回饋的過程，必須以系統生物學觀念，運用高通量檢測及資訊分析等方法來了解基因調控之網絡。在生物學上，微陣列實驗(Microarray) 及其高通量檢測 (high-throughput screen)技術興起，為基因體奠定了一個重要的里程碑。因此，在我們研究當中利用外顯子微陣列晶片(exon array)以及啟動子微陣列晶片 ( promoter array )等高通量基因分析實驗，篩選PC12 (pheochromocytoma cells, PC12) 細胞經由神經生長因子NGF (Nerve growth factor)刺激之後有可能被啟動的啟動子與其下游外顯子基因，兩者結果比較進而深入研究和探討基因分子訊息路徑以及上下游基因之調控機制。經由神經成長因子 (nerve growth factor， NGF) 誘導後分化成類神經細胞並表現出成熟交感神經細胞特性，被稱為 differentiated PC12 細胞，至今已知PC12 細胞株不但作為實驗模型被廣泛用來研究了解神經生理以及神經化學的機制，也是許多研究神經分化和神經凋亡等領域之有效而方便的工具。據文獻指出，當PC12 細胞在正常的生理狀態下其早發性基因(immediate-early gene)的表現量極低，一旦受到壓力、神經傳到物或生長因子(Nerve growth factor, NGF)的刺激後會大量誘發早發性基因的表現，然而神經生長因子NGF 刺激PC12 細胞後在細胞核外的分子訊息傳遞路徑根據文獻已經相當明確，但對於細胞核內分子訊息傳遞路徑至今依然仍須開發。因此，在我們的研究當中，我們以早發性基因c-Fos 基因表現當作活性指標，藉由PC12 細胞經由NGF 刺激短時間之後細胞核內所產生的分子訊號傳遞機制為實驗主軸，探討細胞核內神經生長因子NGF 訊號傳遞的路徑進而將複雜的訊息傳遞路徑配合既有的知識以及未知的新發現構成一個神經細胞分子內網絡(network)。在我們的研究當中，透過詳細完整的實驗的設計得到的實驗結果，再經由 ii Partek 公司提出的分析軟體來偵測顯子微陣列晶片(exon array)以及啟動子微陣列晶片( promoter array )兩種高通量微陣列晶片，並且利用表現平均差異值 (fold-change)和統計檢定的P 值 (P-Values)為篩選方式來篩選有顯著差異的基因。我們將所得到的微陣列晶片的數據進行分析，結果指出在顯子微陣列晶片 (exon array)上依據時間點分析經由生長因子NGF 刺激PC12 細胞0 到180 分鐘後共有8620 個基因通過篩選。在啟動子微陣列晶片( promoter array )上有 24410 個基因通過篩選。根據我們實驗的目的，經由我們實驗的設計後，實驗結果將顯子微陣列晶片(exon array)以及啟動子微陣列晶片( promoter array ) 個別篩選出具有顯著差異的表現基因相互比較，分類基因功能性，並且探討其分子訊息傳遞和上下游基因的調控機制。因此，兩種微陣列基因的比對過後，最終有672 個基因被篩選出分別在生長因子NGF 刺激PC12 細胞之後 60 分鐘及180 分鐘後這些基因皆有表現量。這些基因被發現的功能性大多與神經系統有相關。未來在我們研究當中，希望利用神經因子NGF 刺激PC12 細胞為主軸以高通量微陣列晶片的技術利用系統生物學來整合基因調控元素及轉錄因之間複雜的關係與交互作用。 Gene regulation plays a significant role. The interaction between gene and protein and the transcription of their own genes, as well as the formation of negative feedback loops are the factors to affect gene regulation. Understanding the complexity of such a system is difficult before the invention of microarrays and the high-throughput approaches. Over the years, the application of microarrays and statistical methodologies have become popular in biological research. Studies of biological changes induced by Nerve growth factor (NGF) on PC12 (pheochromocytoma cells) have begun to provide evidence that NGF-related pathways are responsible for signal transduction. In our study, we compared the result of promoter array and exon array database to explore the signal transduction and regulation of upstream and downstream genes in the NGF-signal pathway in PC12 nuclei. Nerve growth factor (NGF) was discovered as a factor for promoting cell growth and cell activation in neural systems. PC12, when treated with NGF, stops dividing and terminally differentiate into neuronal cells. This makes PC12 a good model for studies of neuronal differentiation. Much effort has been done to study the rapid activation in gene transcription following the addition of NGF to PC12. It has been shown in the literature that in a very short time (5-60 minutes) NGF stimulates the transcription of a number of genes, such as c-Fos, which is an immediate early gene known as a regulator for cell differentiation and proliferation in NGF treated systems. There is also some literatures point ing out that although the cellular NGF-signal pathway is much elucidated, less is known about the gene transcriptions in nuclei. In hope of deeper understanding of the molecular mechanisms that underlies the NGF-induced differentiation, we systematically search for iv NGF-responsive immediate-early transcription factors using promoter array and exon array on PC12. The intersection of the two arrays is identified by giving limits to fold-change and P-value in the framework of Partek, a commercial software package. In order to gain insight into the molecular mechanisms that underlies NGF-induced immediate-early gene regulation, we used promoter array and exon array as a tool to search for genes that showed significant changes within 120min NGF treated PC12 cells. To identify genes of interest, we keep genes that showed statistically significant changes from both promoter array and exon array. From the promoter array analysis, we obtained 24410 hits were regulated. Based on the exon array results, we obtained 1546 hits from the 60min NGF treated PC12 cells. After overlapping the hits from both exon array and promoter array, we identified 672 candidate genes that have up-regulated promoter and exon expression. These candidate genes are required to elucidate how these genes function in NGF treated PC12 cells. Our studies demonstrated a high-throughput strategy to study NGF-induced immediate-early gene expression in PC12 cells. The uses of these high-throughput arrays greatly facilitate the study of novel gene regulation and signal transduction occur in PC12 cells.
顯示於類別:	[系統生物與生物資訊研究所] 博碩士論文

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