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研究生：	邱建元 Chiu, Chien-Yuan
論文名稱：	偵測微衛星DNA不穩定雙螢光表現系統的建立 Establishment of a dual-fluorescent MSI reporter system
指導教授：	張玲 Chang, Christina Ling
學位類別：	碩士 Master
系所名稱：	醫學院 - 口腔醫學研究所 Institute of Oral Medicine
論文出版年：	2009
畢業學年度：	97
語文別：	英文
論文頁數：	59
中文關鍵詞：	大腸癌、口腔癌、微衛星不穩定、DNA錯誤配對修復、活性氧分子
外文關鍵詞：	Oral cancer, Colorectal cancer, Microsatellite instability, DNA mismatch repair, Reactive oxygen species (ROS)
相關次數：	點閱：114 下載：2
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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。

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.

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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