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系統識別號 U0026-0812200915394332
論文名稱(中文) 偵測微衛星DNA不穩定雙螢光表現系統的建立
論文名稱(英文) Establishment of a dual-fluorescent MSI reporter system
校院名稱 成功大學
系所名稱(中) 口腔醫學研究所
系所名稱(英) Institute of Oral Medicine
學年度 97
學期 2
出版年 98
研究生(中文) 邱建元
研究生(英文) Chien-Yuan Chiu
學號 T4696401
學位類別 碩士
語文別 英文
論文頁數 59頁
口試委員 指導教授-張玲
口試委員-蔣輯武
口試委員-陳玉玲
口試委員-謝達斌
中文關鍵字 大腸癌  口腔癌  微衛星不穩定  DNA錯誤配對修復  活性氧分子 
英文關鍵字 Oral cancer  Colorectal cancer  Microsatellite instability  DNA mismatch repair  Reactive oxygen species (ROS) 
學科別分類
中文摘要 微衛星DNA是一種由1到6個重複的簡單序列並廣泛的分佈於真核生物基因組中。DNA 錯誤配對修復系統(MMR)的缺失會造成DNA複製錯誤無法修復而產生微衛星DNA不穩定性(MSI)的現象。許多的癌細胞都被發現有MSI的狀態,例如在大腸直腸癌以及口腔癌。而在臨床上,病人呈現MSI將會對化學療法產生抗性。因此,由降低癌症的發生率和致死率的角度癌來看,篩選並建立降低MSI發生率的物質是非常重要的。我已建立帶CA重複序列的微衛星DNA於我們的雙螢光系統表現質體,並轉染到人類的癌症細胞株中。由於氧化壓力會造成MMR系統的不活化,我們已在暫時及穩定轉染的人類大腸癌細胞株中觀察到不同的微衛星序列被氧化壓力誘導產生MSI的靈敏度。藉由流式細胞儀的分析,我們發現以過氧化氫處理的穩定轉染細胞株,其MSI的呈現和過氧化氫的次數和濃度有正向的關係。此外,在口腔癌MSI和抗藥性的關係尚未非常清楚, 在抗藥性測試的結果發現,在兩株口腔癌細胞株OSCC25及CAL27中,OSCC25對藥品的毒性擁有較高的抗性。這個發現符合我們之前對口腔癌細胞MSI狀態的測定。在癌症的預防及控制上,我所建立出來的MSI螢光偵測系統在將來應能應用於抗MSI物質的篩選。
英文摘要 Microsatellites are simple repeats of 1-6 nucleotides, which are widely dispersed in the human genome. Deficiency in DNA mismatch repair (MMR) system results in unrepaired replicative errors preferably in microsatellite sequences, leading to microsatellite instability (MSI). The MSI phenotype has been detected in many other cancers, such as colon and oral cancers. Clinically, cancer patients with MSI develop resistance to chemotherapy. Therefore, it is important to screen and develop anti-MSI compounds in order to reduce cancer incidence and mortality. I have constructed expression plasmids containing (CA)n microsatellite sequences to report the MSI phenotypes in a dual-fluorescence fashion. Since oxidative stress inactivates the MMR function, I have examined the sensitivity of different microsatellites in reporting H2O2–induced MSI frequency in transient and stable transfectants derived from HCT116 and HCT116+chr3 human colorectal cancer cell lines. With flow cytometry, I found that H2O2 increased the MSI frequency in a dose-dependent manner in stable transfectants that harbor (CA)5 and (CA)13 microsatellites, but not in transient transfectants. Furthermore, H2O2 also increased the MSI frequency in a time-dependent manner in the stable transfectants. Since little is known about the correlation between MSI and drug resistance in oral cancer, I tested and found that OSCC25 cells displayed higher drug resistance than CAL27 cells. This is consisted with our previous finding that the MSI frequency is higher in OSCC25 than CAL27 cells. To control and prevent cancer, the dual fluorescent MSI reporters that I have developed will be useful in screening anti-MSI compounds.
論文目次 1. Introduction 1
1.1 Microsatellite and Microsatellite instability 1
1.2 DNA Mismatch Repair System (MMR) 2
1.3 Microsatellite instability in human cancers 4
1.4 Oral cancer 5
1.5 Reactive oxygen species 6
1.6 MSI and drug resistance 7
1.7 Hypothesis 8
1.8 Specific aims 9
2. Materials and methods 10
2.1 Cell lines 10
2.2 Cell culture 10
2.3 Dual Fluorescence MSI Reporter Plasmid construction 11
2.4 Transfection 13
2.5 Stable clone generation 13
2.6 MTT assay 14
2.6.1 Determination the half lethal dose of hydrogen peroxide 15
2.6.2 Drug treatment 15
2.6.3 Determine the toxicity of betel quid ingredients 16
2.7 H2O2 treatment 16
2.8 Flow cytometry 17
2.9 The measurement of MSI frequency 18
2.10 Antioxidant treatment 18
3. Results 19
3.1 Design and construction of the dual fluorescent MSI
reporter plasmid containing the (CA)n microsatellites 19
3.2 Determination of LD50 of H2O2 in the HCT116 human
colorectal cancer cell line 20
3.3 Effects of H2O2 on the MSI frequency in transient
transfectants derived from HCT116 and HCT116+ch3 human
colorectal cancer cell lines 20
3.4 Generation of stable clones harboring dual fluorescence
MSI reporters 21
3.5 Effects of H2O2 on the MSI frequency in HCT116-derived
stable clones 22
3.6 Analysis the alteration of microsatellite sequence of
HCT116 stable clones GFP+ and GFP+/DsRed+ cells 22
3.7 The effects of antioxidant on the HCT116-derived
stable clone 23
3.8 Drug effects on human oral cancer lines 24
3.9 Effects of betel quid ingredients on the cytotoxicity
on human oral cancer cell line 24
4. Discussion 26
5. References 30
6. Tables 35
Table 1. MMR component and functions 35
Table 2. Artificial microsatellite oligonucleotide 36
Table 3. The type of drugs and resistance in MMR deficient cells 36
7. Figures 37
Figure 1. The dual fluorescence MSI reporter constructs
containing (CA)n (n=5, 8, 13) microsatellites. 37
Figure 2. Confirmation of the dual fluorescence MSI reporter
constructs containing the (CA)n microsatellites 38
Figure 3. Determination of LD50 of H2O2 in the HCT116 and
OSCC25 40
Figure 4. Effects of H2O2 on transient transfectants derived
from in HCT116 and HCT116+ch3 human colorectal cancer
cell lines 41
Figure 5. The HCT116 stable clones harbored the dual
fluorescence reporter containing the (CA)5
microsatellite 43
Figure 6. The HCT116 stable clones harbored the dual
fluorescence reporter containing the (CA)13
microsatellite 44
Figure 7. The effect of H2O2 on the stHCT116-(CA)5
stable cell lines 45
Figure 8. The effect of H2O2 on the stHCT116-(CA)13
stable cell lines 47
Figure 9. Direct sequencing of the genomic DNA extracted from
sorted GFP+ or GFP+/DsRed+ cells 49
Figure 10. The effect of antioxidants on the stHCT116
stable cell line 50
Figure 11. Effects of commonly used anti-cancer drugs on
oral cancer cell lines 52
Figure 12. Effects of betel quid ingredients on OSCC25
oral cancer cell line 54
8.Abbreviation list 55
9.Appendix 57
Appendix 1 Frequency of MSI in sporadic cancers 57
Appendix 2 Representative target genes in tumors with MSI-H 58
Appendix 3 The efficacy of chemotherapy drugs in MMR deficient
colorectal cancer 59
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