膀胱癌具有高罹病率及死亡率，是第二常見的生殖泌尿器官腫瘤。找尋非侵入性的膀胱癌生物標記一直是種挑戰。近來，循環系統中的游離DNA被報導用於許多癌症的偵測，但是大多是使用血液樣本。所以，本論文的研究目的，即在於開發與評估使用較不具侵犯性的尿液來做為偵測膀胱癌的工具，特別是利用尿液中游離DNA用於評估做為膀胱癌的診斷標記。研究方向係針對creatinine-adjusted尿液游離DNA濃度、DNA完整性(DNA相對長度)及DNA 修補基因ATM 與CHK2的啟動子高度甲基化等主題進行。首先是creatinine adjusted的尿液游離DNA濃度經由real time PCR定量。在real time PCR定量主要是針對大片斷的游離DNA。結果發現，膀胱癌患者尿液中游離DNA濃度高於沒有罹患任何疾病的人。尿液中游離DNA濃度在36個膀胱癌患者的平均濃度為14.98 pg/

Bladder cancer is the second most common genitourinary tumor and is a significant cause of morbidity and mortality. To find the noninvasive tumor marker for bladder cancer is still a challenge. Recently, the circulating cell-free DNA had been used for detection of some tumors. However, most of these studies selected the blood sample as the free DNA source. The purpose of this thesis is to develop and evaluate the more noninvasive method and tumor marker for bladder cancer detection, especially for the cell-free DNA. Three directions were addressed as followed: 1). urine cell-free DNA makers such as creatinine-adjusted cell-free DNA concentration, 2). DNA integrity (DNA length) and 3). Promoter hypermethylation for DNA repair genes. First, creatinine-adjusted urine cell-free DNA concentration was quantified via real time PCR. The real time PCR-based detection is based on detection large fragment cell-free DNA. Real time PCR-based urine cell-free DNA concentration of bladder cancer patients was higher than controls. Mean concentrations of 36 bladder cancer patients and 93 controls which detected by real time PCR were 14.98 and 1.07 pg/

Contents
Abstract in Chinese…………………………………………………………………… i
Abstract in English……………………………………………………………………. ii
Abbreviations…………………………………………………………………………. iii
1. Introduction………………………………………………………………………… 1
(1) Bladder cancer………………………………………………………………….. 1
(2) Bladder cancer surveillance……………………………………………………. 1
(3) Non-invasive method for bladder cancer detection……………………………. 4
(4) DNA-based molecular marker (cell-free DNA) for cancer detection………….. 4
(5) Free DNA originate from apoptotic or necrotic cells…………………………... 5
(6) Characteristics of apoptosis and necrosis………………………………………. 5
(7) Increased plasma DNA integrity in cancer patients……………………………. 5
(8) Techniques for cell-free DNA quantification…………………………………... 6
(9) Epigenetic………………………………………………………………………. 9
(10) Timing aberrant promoter methylation during tumor progression……………. 9
(11) Promoter hypermethylation as a marker for early tumor detection…………… 9
(12) Aberrant promoter hypermethylation in urine of bladder cancer patients as a non-invasive and sensitive method for cancer detection……………………... 14
(13) DNA damage checkpoints in mammals………………………………………. 14
(14) Ataxia-telangiectasia mutated (ATM)………………………………………… 20
(15) Checkpoint kinase 2 (CHK2)…………………………………………………. 20
(16) Nijmegen breakage syndrome 1 (NBS1)……………………………………... 22
2. Study aims…………………………………………………………………………. 26
3. Materials and Methods…………………………………………………………….. 27
(1) Materials……………………………………………………………………….. 27
A. Real time PCR-based 400 bp DNA quantification…………………………... 27
B. PicoGreen-based urine cell-free DNA quantification………………………... 27
C. DNA integrity analysis via multiplex PCR…………………………………... 27
D. Methylation pattern of DNA repair associated genes was performed via
methylation specific polymerase chain reaction…………………………….. 28
(2) Methods………………………………………………………………………… 29
A. Urine cell-free DNA extraction………………………………………………. 29
B. Calibration……………………………………………………………………. 30
C. Saliva DNA extraction……………………………………………………….. 31
D. Large fragment urine cell-free DNA quantification via Real time PCR……... 32
E. Urine cell-free DNA quantification by PicoGreen…………………………… 36
F. DNA integrity analysis via multiplex PCR…………………………………… 38
G. Urine DNA was modified via sodium bisulfite treatment……………………. 40
H. Methylation profile of DNA repair associated genes was performed via
methylation specific polymerase chain reaction…………………………….. 41
4. Results……………………………………………………………………………… 43
(1) Large fragment urine cell-free DNA quantification via Real time PCR……….. 43
A. Standard curve……………………………………………………………….. 43
B. Large fragment urine cell-free DNA concentration (400 bp) of bladder
cancer patients and controls…………………………………………………... 43
C. Large fragment urine cell-free DNA concentration (400 bp) may act as a
potential marker for bladder cancer patient surveillance……………………. 43
D. Sensitivity and specificity of real time PCR based detection for cell-free
DNA quantification………………………………………………………….. 44
(2) Urine cell-free DNA quantification by PicoGreen……………………………... 44
A. Standard curve………………………………………………………………... 44
B. Urine cell-free DNA concentration of patients with bladder cancer, other
cancer and/or disease and controls………………………………………….. 44
(3) DNA integrity analysis via multiplex PCR…………………………………….. 45
(4) Methylation pattern of ATM, CHK2 and NBS1 genes………………………… 45 A. Methylation pattern of ATM transcription start site in bladder cancer patients 45
B. Methylation pattern of CHK2 transcription start site in bladder cancer patients 45
5. Discussion………………………………………………………………………….. 46
6. References………………………………………………………………………….. 50
7. List of Tables………………………………………………………………………..
Table 1 AJCC 2002 TNM bladder cancer staging………………………………….. 2
Table 2 Techniques that used for DNA methylation detection……………………... 15
Table 3 Methylation of DAPK, RAR

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