Title Page

ABSTRACT

Contents

Chapter 1. Introduction 12

1. Introduction 12

Chapter 2. Materials and Methods 22

2.1. Materials 22

2.2. Cell culture 22

2.3. Optimization of transfection and expression of plasmid 23

2.4. Fluorescence and luminescence measurement of Rluc 8.6-KR 24

2.5. Identification of expression efficiency of pB in cancer cell lines 24

2.6. Cell viability test 24

2.7. Construction of pN-B, pB-M1, and pB-M2 25

2.8. Fluorescence imaging 26

2.9. ROS measurement 27

2.10. Annexin V & PI staining 27

2.11. In vivo test 28

2.12. H&E staining 28

2.13. Statistical analysis 29

Chapter 3. Results and Discussion 30

3.1. Characterization of BRET system 30

3.1.1. Optimization of transfection conditions of pB 30

3.1.2. Observation of BRET phenomenon of Rluc 8.6-KR 30

3.1.3. Transfection and expression efficiency of pB in cancer cell lines 34

3.1.4. BRET-induced cell growth inhibition effect 37

3.2. Construction of subcellular-localized BRET system 37

3.2.1. Cloning of pN-B, pB-M1, and pB-M2 37

3.2.2. Transfection and expression efficiency of pB, pN-B, pB-M1, and pB-M2 38

3.2.3. Subcellular localization of pB, pN-B, pB-M1 and pB-M2 41

3.2.4. Comparison of cell growth inhibition effect of pB, pN-B, pB-M1, and pB-M2 41

3.3. Cell death effect of pB-M1 44

3.3.1. Optimization of transfection and expression efficiency of pB-M1 44

3.3.2. BRET-induced cell death of pB-M1 44

3.3.3. BRET-induced ROS generation of pB-M1 46

3.3.4. Tumor growth inhibition effect of pB-M1 in vivo 50

3.3.5. Evaluation of toxicity of Co-h in vivo 50

Chapter 4. Conclusion 55

References 57

국문초록 64

Figure 1.1. Mechanism of PDT. 14

Figure 1.2. Absorption and emission spectrum of Rluc 8.6-KR. 17

Figure 1.3. Bioluminescence resonance energy transfer (BRET) system. 18

Figure 1.4. Scheme of subcellular-localized BRET system for enhanced PDT. 20

Figure 3.1. Optimization of transfection conditions of pB in HEK293T. 31

Figure 3.2. Expression of Rluc 8.6-KR by time. 32

Figure 3.3. Identification of BRET between Rluc 8.6 and KR. 33

Figure 3.4. Identification of transfection and expression efficiency of pB in cancer cell lines. 35

Figure 3.5. Identification of BRET-induced cell growth inhibition effect of Rluc 8.6-KR. 36

Figure 3.6. Construction of pN-B, pB-M1, and pB-M2. 39

Figure 3.7. Identification of transfection and expression efficiency of pB, pN-B, pB-M1, and pB-M2 in B16F10. 40

Figure 3.8. Observation of subcellular localization of pB, pN-B, pB-M1, and pB-M2. 42

Figure 3.9. Comparison of cell growth inhibition effect of pB, pN-B, pB-M1, and pB-M2. 43

Figure 3.10. Re-optimization of transfection conditions about pB-M1 and B16F10 cell. 45

Figure 3.11. Cell growth inhibition effect of pB-M1 by Co-h concentration. 47

Figure 3.12. Identification of BRET-induced cell death of pB-M1. 48

Figure 3.13. BRET-induced ROS generation of pB-M1. 49

Figure 3.14. Tumor growth inhibition effect of pB-M1. 52

Figure 3.15. Expression of pB-M1 in vivo. 53

Figure 3.16. Evaluation of toxicity of Co-h in vivo. 54