臺灣博碩士論文加值系統

Acknowledgment i
Abstract (Chinese) ii
Abstract iii
Table of Contents iv
List of Figures viii
List of Tables x
Introduction 1
1. Radiotherapy and Tumor Monitoring 1
2. Liquid Biopsy 2
3. Exogenous Synthetic Biomarkers 4
4. Secreted Reporters 5
4.1 Human Chorionic Gonadotropin (hCG) 5
4.2 Gaussia Luciferase (GLuc) 6
5. Radiation-Responsive Signaling Pathways 6
5.1 TGF-β/Smad Pathway 7
5.2 NRF2/Antioxidant Response Element (ARE) Pathway 8
5.3 EGR-1/Serum Response Elements (SRE) Pathway 9
6. Prostate Cancer 9
Materials and Methods 12
1. Cell Lines 12
2. Western Blotting 12
3. Cytotoxicity of Ionizing Radiation (MTT Assay and CCK-8 Assay) 13
4. Irradiation Procedure 14
5. Transient Transfection 14
6. IR-Responsive Plasmids Construction 15
7. Establishment of IR-Responsive Stable Clones 16
8. Luciferase Assay 17
9. ELISA Enzyme-linked immunosorbent assay (ELISA) 19
10. Establish IR-Resistant Prostate Cancer Cell Lines 20
11. Statistics 20
Results 21
1. Ionizing radiation activates radiation-responsive protein expressions in prostate cancer cells. 21
2. Cell viabilities of prostate cancer cells are decreased in a dose-dependent manner after IR treatment. 23
3. Smad binding elements (SBE) can function as an IR-responsive promoter. 24
4. Establishment of constructs encoding the secreted reporter hCG and GLuc driven by the IR-responsive promoter. 25
5. IR-responsive constructs can respond to IR, and the resulting signals could correlate with the IR-induced cytotoxicity effect in 293FT cells. 26
6. IR-responsive prostate cancer cells exhibit a high correlation between secreted biomarker levels and RT outcomes. 27
7. IR-resistant prostate cancer cells respond to IR differently compared to parental prostate cancer cells, as shown in IR-responsive protein expressions. 29
Discussion 30
Conclusions 38
References 39
Figures 52
Tables 74
Appendices 78
1. Cell Lines 78
2. Cell Culture 78
3. Chemicals, Reagents, and Antibodies 78
4. Equipment 80
5. Software 81
Supplementary 1. Mechanism of IR in TGF-β/Smad signaling pathway. IR-inducing DNA double-strand breaks are detected by ATM activation. 82
Supplementary 2. Mechanism of IR in NRF2/Antioxidant Response Element (ARE) signaling pathway. 83
Supplementary 3. Mechanism of IR in EGR-1/Serum Response Elements (SRE) signaling pathway. 84
Supplementary 4. Mechanisms of ROS production, and cellular response to ROS in prostate cells. 85
Supplementary 5. Crosstalk between TGF-β and ROS. 86
Supplementary 6. Transcriptional activities of NF-κB-Luc2P and E9-FLuc 2 to 24 hours after single-dose irradiation. 87
Supplementary 7. Strategies of using genetically encoded synthetic biomarkers leverage tumor-specificity to achieve detectable signals. 88
Supplementary 8. Vector map of the pCDH-EF1S-hCG-P2A-copGFP-T2A-puro with agarose gel electrophoresis to confirm the production of the constructed products. 89
Supplementary 9. Vector map of the pCDH-EF1S-hCG-P2A-Gluc-T2A-puro with agarose gel electrophoresis to confirm the production of the constructed products. 90
Supplementary 10. Vector map of the pCDH-EF1S-Nluc-P2A-mCherryT2A-puro with agarose gel electrophoresis to confirm the production of the constructed products. 91

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