臺灣博碩士論文加值系統

缺氧(Hypoxia)意指細胞環境氧氣下降。缺乏氧氣時，細胞內的缺氧誘導因子-1a會與缺氧誘導因子-1b形成二聚體，並結合到DNA序列上的缺氧反應組，進而調控下游基因表現。受到缺氧調控的基因已經被證實參予在細胞增生，血管新生，醣解作用，細胞凋亡及腫瘤轉型等生物近程中。缺氧反應組包含一核心序列“RCGTG”，除了此核心序列外，缺氧誘導因子-1對缺氧反應組的親和性也受變異性較大的旁側序列影響。本論文研究旨在利用較具彈性的搜尋模型尋找基因體中受缺氧誘導因子-1調控之基因。模型的建立是將二十條已知缺氧反應組的序列並排，並將並排之序列以隱藏式馬可夫鍊的理論基礎建立搜尋模型。藉由該模型搜尋人類及小鼠之基因促進子，共有8170個人類及6477個小鼠的候選基因可在截止分數（cutoff score）為-1.8時被辨認出來。大約三分之一的候選基因可同時在人類和小鼠的基因體中被辨識。分析以去鐵劑DFO模擬的缺氧處理時，亂數抽樣的50個候選基因表現情形，發現正分(positive score) 候選基因的調控比率為91%。由此結果可知約有2500個人類基因和1600個小鼠基因的表達可能受到缺氧誘導因子-1的調控。分析候選基因的調控模式，發現56%的候選基因在不同細胞株中有相同的調控結果。進一步分析發現候選基因受調控的時間點可細分為早期調控，延遲調控及二相(biphasic)調控。在以染色質免疫沉澱技術偵測缺氧誘導因子-1與候選基因之缺氧反應組結合情形的實驗中，發現缺氧情形下候選基因表現量的改變與細胞核內的缺氧誘導因子-1的總量及其對DNA的結合能力有相關性。綜合以上結果顯示，利用隱藏式馬可夫鍊為理論基礎的搜尋模型可以有效地大量尋找特定轉錄因子所調控的基因表現。

Hypoxia is the reduction of environmental oxygen In absent of oxygen, hypoxia inducible factor-1 a (HIF-1a) dimerizes with HIF-1b and binds to the hypoxia response element (HRE) on the target DNA sequence. HIF-1a regulated genes have been found to be involved in cell proliferation, angiogenesis, glycolysis, apoptosis, and tumor formation. The HRE with a short core sequence “RCGTG” is necessary but not sufficient to be bound by HIF-1a. The flanking region also determines the binding activity. Accordingly, the 20 well known HREs were retrieved, aligned, and built up a hidden Markov model based HRE profile. The HMM-based HRE profile was used to search candidate HRE on the promoter region of human and mouse genes. 8170 human genes and 6477 mouse genes were identified by the cutoff score -1.8. About one-third of putative these HIF-1a regulated genes are conserved between human and mouse genome. The expression profiles of randomly picked fifty genes were investigated at various time points after DFO mimic hypoxia treatment. The regulation rate of the genes with positive score is 91%. This indicates that about 2500 human genes and 1600 mouse genes could be regulated by HIF-1a. In analysis of regulation pattern the candidate genes were regulated consistently among different cells or were specifically expressed and/ or regulated in one cell. For the time course analysis, the genes regulated by hypoxia can be further classified into one of early, delay, or biphasic category. The regulation patterns are similar in hypoxia and DFO treatment suggesting that DFO is a proper hypoxia mimetic. By detecting intra nuclear HIF-1a protein and in vivo binding of HIF-1a on the candidate HRE, it was demonstrated the altered RNA expression in candidate genes under chemical or true hypoxia is correlated with nuclear HIF-1a protein level and the binding activity. Put all together, this study demonstrated a high throughput screening and verification approach in understanding the whole picture of gene regulation mediated by hypoxia.

ACKNOWLEDGMENTS I
TABLE OF CONTENT II
FIGURES AND TABLES IV
ABSTRACT IN CHINESE 1
ABSTRACT 2
ABBREVIATIONS 3
INTRODUCTION 6
MATERIALS AND METHODS 11
CELL CULTURE AND HYPOXIA TREATMENT 11
Cell maintenance and subculture 11
Hypoxia treatment 11
ISOLATION OF TOTAL RNA 12
ISOLATION OF GENOMIC DNA 13
SOFTWARE INSTALLATION AND USAGE 13
PROMOTER SEQUENCE RETRIEVAL 14
PATTERN SEARCH OF PROMOTER SEQUENCES 14
MODEL CONSTRUCTION 14
a. HREs retrival 14
b. Multiple sequence alignment 15
c. Construction of Hidden Markov Model 15
DATABASE 16
MULTIPLEX RT-PCR METHODOLOGY 17
a. Reverse Transcription-Polymerase Chain Reaction 17
b. Exon sequence retrieval and optimization of Multiplex PCR 18
QUANTIFICATION OF THE CANDIDATE GENE EXPRESSION BY REAL-TIME PCR 19
ISOLATION OF NUCLEAR PROTEINS 20
IMMUNO-BLOTTING 20
CHROMATIN IMMUNOPRECIPITATION (CHIP) 21
RESULTS 23
HMM PROFILE CONSTRUCT OF HRE 23
HRE MODEL SEARCH ON KNOWN HIF-1�� REGULATED GENES 23
GENOME-WILD SEARCH BY HRE MODEL 24
COMPARATIVE STUDY OF THE HITS FOUND IN HUMAN AND MOUSE 24
VERIFICATION OF HMM-IDENTIFIED CANDIDATE GENES 25
Strategy of high-throughput validation of candidate gene expression 25
Candidate gene expression under chemical hypoxia 26
Regulation rate of candidate genes under chemical hypoxia 26
Cell type specific regulation hypoxia targeted genes 27
Time-dependent regulation of candidate genes by hypoxia 27
QUANTIFICATION OF CANDIDATE GENE EXPRESSION UNDER TRUE HYPOXIA 28
UP-REGULATION OF HIF-1�� PROTEIN UNDER CHEMICAL AND TRUE HYPOXIA 29
IN VIVO BINDING OF HIF-1���nON THE REGULATED PROMOTER OF CANDIDATE GENES 29
REFERENCE 71
APPENDIX 78

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