博碩士論文 962211010 詳細資訊




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姓名 邱佩洵(Pei-hsun chiu)  查詢紙本館藏   畢業系所 系統生物與生物資訊研究所
論文名稱 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 identifygenome-wide regulatory elements and genetranscription in the neuron-differentiation ofPC12 cell line )
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摘要(中) 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。
摘要(英) 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.
關鍵字(中) ★ 細胞 神經生長因子 微陣列 關鍵字(英) ★ PC12 NGF microarray
論文目次 Chinese Abstract ----------------------------------------------------------------------------- i
English Abstract ------------------------------------------------------------------------------iii
Acknowledgments----------------------------------------------------------------------------v
Table of contents -----------------------------------------------------------------------------vi
Index of figure---------------------------------------------------------------------------------viii
Index of Table----------------------------------------------------------------------------------ix
Abbreviations-----------------------------------------------------------------------------------x
一、 Introduction-----------------------------------------------------------------------------1
二、 Material and Method----------------------------------------------------------------10
2.1 Cell culture-----------------------------------------------------------------------10
2.2 Cell survival assay--------------------------------------------------------------11
2.3 Cell cultured plate coating----------------------------------------------------11
2.4 Cell differentiation assay------------------------------------------------------11
2.5 Defined neuronal differentiation of PC-12 neurite----------------------12
2.6 RNA preparation----------------------------------------------------------------12
2.7 cDNA synthesis-----------------------------------------------------------------13
2.8 Real time PCR------------------------------------------------------------------14
2.9 Immunostaining-----------------------------------------------------------------14
2.10 Nuclear protein extraction and DNase I digestion flow chart-----15
2.11 Micorarray classification----------------------------------------------------17
2.11.1 Exon array GeneChip® Rat Exon 1.0 ST-------------------------17
2.11.2 Mouse Promoter 1.0R Array------------------------------------------18
2.12. Microarray hybridization and statistical analysis---------------------19
三、Results-------------------------------------------------------------------------------------21
vii
3.1 NGF-induced PC12 model description---------------------------------------21
3.1.1 Induction of neurite outgrowth through our model in PC12 cells
3.2. Validation experiment prior to microarray hybridization----------------22
3.2.1 Exon array sample prepared-------------------------------------------22
3.2.2 Promoter array sample prepared-------------------------------------22
3.3. Microarray quality and analysis using parket------------------------------23
3.3.1 partek screener solution-------------------------------------------------23
3.3.2 Exon array------------------------------------------------------------------23
3.3.3 Promoter array-------------------------------------------------------------24
3.3.4 Identify the overlap of the two gene lists----------------------------25
四、Discussion---------------------------------------------------------------------------------26
五、Conclusion---------------------------------------------------------------------------------29
六、Reference----------------------------------------------------------------------------------30
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指導教授 吳立青、凌慶東
(Lei-Chin Wu、Qing-Dong Ling)
審核日期 2009-7-2
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