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Title Page
ABSTRACT
Contents
Chapter 1. Introduction 13
Chapter 2. MATERIALS and METHODS 32
2.1. Materials 32
2.1.1. Cell lines, culture media, and culture conditions 32
2.1.2. Exosome free FBS preparation 32
2.1.3. Molecular beacon 33
2.1.4. Urine collection 33
2.2. Isolation of exosome 33
2.2.1. Isolation of exosome from cell culture by ExoQuick-TC™ 34
2.2.2. Isolation of exosome from cell culture medium by ultracentrifugation 34
2.2.3. Isolation of exosome from human urine by ultracentrifugation 34
2.3. Characterization of isolated exosome from cell lines 35
2.3.1. Transmission electron microscopy (TEM) analysis 35
2.3.2. Nanoparticle Tracking Analysis 35
2.4. Characterization of isolated exosomes from human urine 35
2.4.1. Transmission electron microscopy analysis 36
2.4.2. Dynamic light scattering analysis (DLS) 36
2.4.3. Western blot analysis 36
2.5. Quantification of purified exosomes 37
2.5.1. Bicinchoninic acid assay (BCA assay) 37
2.5.2. Exosome quantitation assay (EXOCET) 37
2.6. Real-time PCR analysis of miRNA in cells and exosomes 38
2.6.1. RNA isolation 38
2.6.2. cDNA synthesis 38
2.6.3. Real-time PCR analysis 39
2.7. Detection of miRNA using MB 39
2.7.1. In situ detection of miRNA in solution using MB 39
2.7.2. In situ detection of miRNA in exosome from prostate cancer cells using MBs 40
2.7.3. Cross hybridization reaction for dual detection of miRNA using MBs in solution 40
2.7.4. Dual detection of miRNAs using MBs in solution 40
2.7.5. Dual detection of miRNAs using MBs in exosome 40
2.7.6. In situ detection of miRNA in exosome from human urine using MB 41
2.8. Statistical analysis 41
Chapter 3. RESULTS and DISCISSION 42
3.1 Detection of miRNA in exosome of cell culture using MB 42
3.1.1. In situ detection of miRNA in solution using MB 42
3.1.2. Production, isolation, and characterization of exosomes from prostate cancer and prostate non-cancer cells 42
3.1.3. Real-time PCR analysis of miRNA in cells and exosomes 44
3.1.4. In situ detection of miRNA in exosome from prostate cancer cells using MBs 44
3.1.5. Detection of miRNAs in exosomes isolated by ultracentrifugation 49
3.1.6. Dual detection of miRNAs using MBs in solution 51
3.1.7. Dual detection of miRNA in exosome from prostate cancer cells using MBs 56
3.2. Detection of miRNA using MB in exosomes from human urine 58
3.2.1. Isolation and characterization of exosomes from human urine 58
3.2.2. Quantification of amount of exosome particles and concentration of proteins 58
3.2.3. Detection of miR-21 using MB in solution 63
3.2.4. In situ detection of miR-21 in exosome from human urine using MB 63
Chapter 4. CONCLUSIONS 69
참고문헌 71
국문초록 76
Curriculum Vitae 80
Table 1. Potential miRNA biomarkers associated with circulating vesicles in PCa 24
Table 2. The prostate cancer and prostate non-cancer cell lines 30
Table 3. The molecular beacon designs and their target miRNAs 31
Table 4. Information of exosome isolated from human urine 65
Figure 1. Biogenesis of exosomes. Exosomes originate from the multivesicular bodies (MVBs)... 15
Figure 2. Typical structure and composition of exosomes. Exosomes are surrounded by a... 17
Figure 3. The synthesis of miRNA. The synthesis of miRNA starts from the nucleus when the... 19
Figure 4. Putative sources of circulating microRNAs (miR/miRNA). MiRNA in circulation may... 20
Figure 5. Limitations of current exosome miRNA detection method. PCR-based method is... 25
Figure 6. Molecular beacon (MB). MB is a nano-sized oligonucleotide molecule and a dual-... 28
Figure 7. Illustration for the in situ detection of exosomal miRNA using MB for the diagnosis of... 29
Figure 8. Detection of miR-375 and miR-574-3p using MB in solution 43
Figure 9. Characterization of exosomes from prostate cancer cells by electron microscopy.... 46
Figure 10. The analysis of cellular and exosomal miRNAs using a real-time PCR analysis.... 47
Figure 11. Detection of miR-375 and miR-574-3p using MB in exosomes of PC-3 and DU145... 48
Figure 12. Comparison of miR-375 and miR-574-3p expression level to RWPE-1 cells. The... 50
Figure 13. Detection of miR-375 and miR-574-3p in exosome purified by ExoQuick... 53
Figure 14. The cross reaction of miR-375 or miR-574-3p with their targeting MBs in solution.... 54
Figure 15. Dual detection of miR-375 and miR-574-3p using MBs in solution. One hundred nM... 55
Figure 16. Dual detection of miR-375 and miR-574-3p using MBs in exosome from prostate... 57
Figure 17. Characterization of human urinary exosomes by electron microscopy. Exosomes... 60
Figure 18. Size distribution analysis of human urinary exosomes extracted for... 61
Figure 19. Characterization of human urinary exosomes by Western blot. Western blot analysis... 62
Figure 20. Detection of miR-21 using MB in solution. MB-21 was hybridized with different... 66
Figure 21. Detection of miR-21 from exosomes isolated from human urine. 67
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