紫外光照射人類胚胎纖維母細胞之轉錄體分析

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研究生：	張昆鈺 Kun-Yu Chang
論文名稱：	紫外光照射人類胚胎纖維母細胞之轉錄體分析 Transcriptomic Analysis of Ultraviolet B Response in Human Embryonic Lung Fibroblast
指導教授：	葉世榮 S. R. Yeh 許志楧 Ian C. Hsu
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2006
畢業學年度：	94
語文別：	英文
論文頁數：	49
中文關鍵詞：	基因微陣列、紫外光、人類胚胎纖維母細胞、轉錄體、粒腺體、呼吸鍊
外文關鍵詞：	cDNA microarray, Ultraviolet, Human Embryonic Lung Fibroblast, transcriptomic, mitochondria, respiratory chain
相關次數：	點閱：3 下載：0
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UVB is a well-known environmental carcinogen, triggering cell cycle arrest and regulating metabolic activities. To investigate the transcriptional time course responses to UVB radiation, we performed loop-designed cDNA microarray experiments with D-optimal Algorithm analysis to evaluate the relative gene expressions of normal human fibroblasts. Functional classification implied the regulation of suggested biological responses to UVB radiation at the cellular level. The suggested biological responses revealed that transcriptional and translational activities were regulated without bias to activation or repression, inflammatory responses were induced, transient cell cycle arrest occurred in early stage, DNA replication was gradually elevated, and energy production was promoted. In general, common gene regulations altered by UVB were obviously shown in this study. Interestingly, our data showed that cells irradiated by UVB exhibited prolonged transcriptional activation of mitochondrial respiratory chain. The increase of ratio of relative mitochondrail activity also manifested that relative mitochondrial activity was induced by UVB irradiation. Here, our data demonstrates that UVB provokes the prolonged transcriptional activation of mitochondrial respiratory chain.

Contents
ABSTRACT    I
CONTENTS    II
FIGURE LIST    IV
TABLE LIST    VI
CHAPTER 1 INTRODUCTION    1
1.1     PAPER REVIEW    1
1.1.1    DNA Damage and DNA Repair    3
1.1.2    Stress Signaling Pathways    3
1.1.3    Apoptosis    4
1.1.4    Immunoregulation    5
1.2     MOTIVATION    5
CHAPTER 2 MATERIAL AND METHOD    7
2.1     CELL CULTURE    7
2.2     UVB IRRADIATION SYSTEM    8
2.2.1    UVB Lamp    8
2.2.2    View Cabinet    8
2.2.3     UV Curing Radiometer    9
2.3     MTT ASSAY    9
2.4     CELL SURVIVAL ASSAY    12
2.5     CDNA MICROARRAY SYSTEM    14
2.5.1    Printing System    14
2.5.2    Laser Scanning System    14
2.5.3    cDNA Probe    15
2.5.4    Slide    15
2.6     MICROARRAY ANALYSIS    16
2.6.1    The Loop Design of cDNA Microarray Experiment    16
2.6.2     Target preparation    17
2.6.3    Microarray Hybridization    18
2.6.4    Data Acquisition and Data Preprocess    18
2.6.5    Functional Classification    20
2.7    REAL-TIME PCR    20
CHAPTER 3 RESULTS    22
3.1     MICROARRAY PREPROCESS    22
3.1.1    MA plot of Microarray experiment    22
3.1.2    Parameter Evaluation of Clones    26
3.1.3    Functional Classification    27
3.1.4    Activation of Mitochondrial Respiratory Chain    28
3.2     MTT ASSAY    29
3.3     CELL SURVIVAL ASSAY    31
3.4     RELATIVE MITOCHONDRIAL ACTIVITY    32
3.5     REAL-TIME PCR    33
CHAPTER 4 DISCUSSION    35
4.1     CELL ADHESION AND MOTILITY    35
4.2     STRESS AND IMMUNE RESPONSE    36
4.3     CELL SURVIVAL AND DEATH    37
4.4     RESPIRATORY CHAIN    38
4.5     TRANSCRIPTION AND TRANSLATION    40
4.6     METABOLISM AND TRANSPORT    41
4.7     SIGNAL TRANSDUCTION    42
4.8     CONCLUSION    42
REFERENCES    44

Figure List
Figure 1.1 Effective erythermal action spectrum    2
Figure 2.1 Appearance of the UVB lamp with LONGLIFE filter assembly, EB-160C     8
Figure 2.2 UVB bulb spectrum (redrawn)    8
Figure 2.3 View Cabinet CM-10    9
Figure 2.4 UV curing radiometer (International Light)    9
Figure 2.5 Loop design of 10 microarray experiments    17
Figure 3.1 The MA-plot of Mock experiment    23
Figure 3.2 (a) The MA-plot of Slide M01 (4Cà4H) presents the control sample at 4 hour hybridized with the UVB irradiated sample at 4 hour    24
Figure 3.2 (b) The MA-plot of Slide M02 (4Hà8C) presents the UVB irradiated sample at 4 hour hybridized with the control sample at 8 hour    24
Figure 3.2 (c) The MA-plot of Slide M03 (8Cà8H) presents the control sample at 8 hour hybridized with the UVB irradiated sample at 8 hour    24
Figure 3.2 (d) The MA-plot of Slide M04 (8Hà16C) presents the UVB irradiated sample at 8 hour hybridized with the control sample at 16 hour    24
Figure 3.2 (e) The MA-plot of Slide M05 (16Cà16H) presents the control sample at 16 hour hybridized with the UVB irradiated sample at 16 hour    25
Figure 3.2 (f) The MA-plot of Slide M06 (16Hà24C) presents the UVB irradiated sample at 16 hour hybridized with the control sample at 24 hour    25
Figure 3.2 (g) The MA-plot of Slide M07 (24Cà24H) presents the control sample at 24 hour hybridized with the UVB irradiated sample at 24 hour    25
Figure 3.2 (h) The MA-plot of Slide M08 (24Hà4C) presents the UVB irradiated sample at 24 hour hybridized with the control sample at 4 hour    25
Figure 3.2 (i) The MA-plot of Slide M09 (8Hà24H) presents the UVB irradiated sample at 4 hour hybridized with the UVB irradiated sample at 8 hour    26
Figure 3.2 (j) The MA-plot of Slide M10 (16Hà4H) presents the UVB irradiated sample at 16 hour hybridized with the UVB treated sample sample at 4 hour    26
Figure 3.3 The distribution of the parameter γ with the median of 0.00128    26
Figure 3.4 The distribution of the parameter σ with the median of 0.20687    27
Figure 3.5 Gene expression profiles of sixteen regulated genes involved in the respiratory chain    29
Figure 3.6 The MTT assay of serial dose of UVB irradiation    30
Figure 3.7 The MTT assay of time-course of UVB irradiation    31
Figure 3.8 Survival fraction of time-course experiment of 600J/m2 UVB irradiation     32
Figure 3.9 (a) Surviving fraction and relative MTT OD ratio    33
Figure 3.9 (b) Relative mitochondrial activity    33
Figure 3.10 Correlation of real-time PCR data and microarray data    34
Figure 4.1 Five complexes involved in respiratory chain in mitochondria    39


Table List
Table 1 Primer list for real-time PCR analysis of selected genes    21
Table 2 Number of genes classified at the four time points in the nine categories    28

                                

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