Title Page
Contents
Abbreviations and Symbols 11
Abstract 13
I. Introduction 16
1. Liver cancer 16
1) Hepatocellular carcinoma 16
2) Alpha-fetoprotein 18
3) Imaging in clinical finding for diagnosis and follow-up 21
2. Tumor-developed experimental animal model 22
1) Tumor-cell implantation models 22
2) Chemically induced HCC models 23
3) Genetically engineered mouse - Transgenic animals 25
3. Multimodal Imaging 30
1) Optical imaging 30
2) PET, CT, and PET/CT 32
3) MRI 35
4) Advantages of multimodal imaging 36
5) Imaging models (reporter animal) 36
4. Purpose of this study 39
II. Materials and Methods 40
1. Cell lines and AFP secretion level test 40
2. Reporter vector construction 40
3. Transient transfection and transfection efficient analysis 41
4. Generation of transgenic mice 42
5. RT-PCR 42
6. In vivo bioluminescence imaging 43
7. AFP expression in neonatal liver 44
8. HCC-induced murine model 45
9. The liver specific-contrast enhanced-CT images acquisition 46
10. MRI acquisition 46
11. 18F-FDG-PET/CT images acquisition(이미지참조) 47
12. Histologic analysis 47
III. Results 49
1. In vivo studies 49
2. Production of transgenic mice 49
3. Confirmation of reporter gene expression in transgenic mice 52
4. Correlation with the AFP expression level and fLuc activity in neonatal transgenic mice 56
5. Confirmation of HCC development in DEN-model 57
6. MRI and 18F-FDG-PET/CT imaging of HCC-bearing model(이미지참조) 59
7. Longitudinal monitoring of HCC in HBx-Tg models with 18F-FDG PET/CT (이미지참조) 65
8. Verification of DEN-induced HCC in AFPfLuc-Tg model 65
9. Histopathology 70
IV. Discussion 74
References 79
국문초록 91
Table 1. Serum Markers for HCC 19
Table 2. Summary of Chemically Induced HCC-models 24
Table 3. Summary of Transgenic Mouse Models for HCC 29
Table 4. Overview of mouse imaging systems 33
Table 5. In vitro AFP secretion level test by radioimmunoassay 50
Table 6. Summary of HCC Models by DEN Treatment and Oncogene Expression 64
Fig. 1. The cancer incidence rates at Korea (2004-2008). 17
Fig. 2. Concentration of AFP and albumin mRNA were determined by dot blot hybridization at different stages before and after birth. Percentage of labeled nuclei after a 12-hr pulse of [³H]thymidine was determined in... 20
Fig. 3. Current model of sex hormone receptor-dependent HCC development in DEN-induced hepatocarcinogenesis. AR showed the gender difference in HCC incidence.... 26
Fig. 4. Multimodal imaging systems in clinical and preclinical trials and acquired images. 31
Fig. 5. Interested tumor targeted imaging using tumor-specific enhancer/promoter system in BLI. 34
Fig. 6. Transgenic mice for liver cancer. A and B was histologic analysis by H&E staining from HBx-transgenic mouse and Alb/c-myc transgenic mouse, respectively.... 38
Fig. 7. Scheme for production of transgenic mice. The transgene confirmation was performed genomic PCR and expression and its rate of transgene was verified by BLI. 43
Fig. 8. Relative fLuc activities of HuH-7 and 293A transfected with pAFP-fLuc compared to those of trasnfection with pCMV-fLuc. 51
Fig. 9. Transgene confirmation by genomic PCR. Eleven trasngene-carring mice were finally selected among 110 founders. There were eight lines of male transgenic mice (no. 39, 42, 57, 59, 74, 78, 81, and 82) and 3... 53
Fig. 10. BLIs for the neonates of wild-type and four positive founders. Each positive founder was mated with the wild-type female or male partner.... 54
Fig. 11. Confirmation of the BL signal and fLuc expression in the mRNA level of the liver from transgenic mice. 55
Fig. 12. BLIs of the neonates according to time after birth. BLIs of the neonates from positive founder 39 were obtained 1, 5, and 10 days after birth.... 57
Fig. 13. Relationship between the AFP expression level and fLuc activity in neonatal liver. After BLIs of the neonates from 1 to 10 days after birth were acquired, RT-PCR for AFP transcripts (A) and western blot... 58
Fig. 14. Confirmation of chemically induced HCC by DEN with MRI (A) and CT images (B and C) using liver-specific contrast agent. The images was acquired at 33 weeks after DEN injection, and tumors were... 60
Fig. 15. Verification of AFP secretion (A) and DEN induced HCC by H&E staining (B). 61
Fig. 16. Representative coronal MRI and 18F-FDG PET/CT images in the DEN-model. After acquisition of MRI with T2-weighted sequence, PET/CT images were obtained with 18F-FDG....(이미지참조) 62
Fig. 17. Representative axial MRI and 18F-FDG PET/CT images in HBx-Tg model 11 months after birth. After acquisition of MRI with T2-weighted sequence, PET/CT images were obtained with...(이미지참조) 63
Fig. 18. Longitudinal monitoring of HCC in HBx-Tg model according to turn or growth over time. The HBx-Tg mice were serially imageg after birth 11 to 20 months by PET/CT with 18F-FDG....(이미지참조) 66
Fig. 19. MRI and ex vivo BLI of DEN-induced HCC in AFPfLuc-Tg models. DEN-induced AFPfLuc-Tg model was scanned with 3.0T MRI (A) The ex vivo BLI of same DEN-induced AFPfLuc-Tg model was... 67
Fig. 20. Longitudinally monitoring of hepatocarcinogenesis by BLIs of DEN-induced HCC in AFPfLuc Tg model. Eight months after treatment with DEN, the BLIs of the transgenic mouse were acquired for 6... 68
Fig. 21. Photon efflux according to time progression in DEN-induced AFPfLuc-Tg models. 69
Fig. 22. Histologic validation of the liver in normal mouse (A) DEN-model (B) HBx-Tg model (C) stained with H&E. Note that tumor nodules are either encapsulated (B, arrows) or diffused (C, asterisk)... 71
Fig. 23. Histological assessment of DEN-induced HCC in AFPfLuc-Tg model. H&E staining (A) and IHC with the AFP antibody (B) of the liver specimen of the DEN-treated AFPfLuc-Tg models were performed. 72