Title Page
ABSTRACT
Contents
INTRODUCTION 15
MATERIALS AND METHODS 22
1. 2D cell culture 22
2. 3D cell culture 22
3. Immunofluorescence staining 23
4. RNA extraction and cDNA synthesis 24
5. RT-PCR 25
6. Quantitative RT-PCR 25
7. 17β-estradiol (E2) and dihydrotestosterone (DHT) treatment 26
8. Macrophage differentiation and cytokine treatment 27
9. Direct co-culture of macrophage and prostate cells 27
10. Indirect co-culture of macrophage and prostate cells 27
11. Cytokine array 28
12. Statistical analysis 28
RESULTS 29
1. Establishment of 2D and 3D co-culture system for RWPE-1 and WPMY-1 cells 29
2. Localization of prostate epithelial and stromal cells in 2D and 3D co-culture system. 32
3. Effect of 17β-estradiol and dihydrotestosterone on cell proliferation of 2D and 3D co-cultured prostate cells. 35
4. Effect of 17β-estradiol and dihydrotestosterone on expression of estrogen receptor and androgen receptor in 2D and 3D co-cultured prostate cells. 39
5. Induction of macrophage differentiation by proinflammatory cytokines. 42
6. Effect of macrophage on fibrogenesis in RWPE-1 and WPMY-1 cells. 47
7. Effects of 17β-estradiol and dihydrotestosterone on fibrosis in prostate cells. 50
8. Effects of 17β-estradiol and dihydrotestosterone on fibrosis of prostate cells indirectly co-cultured with M2 macrophages. 52
9. Identification of cytokines secreted by macrophage. 56
DISCUSSION 59
REFERENCES 66
국문요약 81
Figure 1. Establishment of 2D co-culture system for RWPE-1 and WPMY-1 cells. 30
Figure 2. Establishment of 3D co-culture system for RWPE-1 and WPMY-1 cells. 31
Figure 3. Localization of CK18 and vimentin in 2D co-cultured RWPE-1 and WPMY-1 cells. 33
Figure 4. Localization of CK18 and vimentin in 3D co-cultured RWPE-1 and WPMY-1 cells. 34
Figure 5. Expression of estrogen receptor (ER) and androgen receptor (AR) in RWPE-1 and WPMY-1 cells. 36
Figure 6. Effects of 17β-estradiol and dihydrotestosterone on the proliferation of 2D co-cultured RWPE-1 and WPMY-1 cells. 37
Figure 7. Effects of 17β-estradiol and dihydrotestosterone on the proliferation of 3D co-cultured RWPE-1 and WPMY-1 cells. 38
Figure 8. Effects of 17β-estradiol and dihydrotestosterone on expression of estrogen receptor and androgen receptor in 2D co-cultured RWPE-1 and... 40
Figure 9. Effects of 17β-estradiol and dihydrotestosterone on expression of estrogen receptor and androgen receptor in 3D co-cultured RWPE-1 and... 41
Figure 10. Morphology of differentiated THP-1 cells after cytokine treatment. 43
Figure 11. Expression of M1 and M2 macrophage marker genes in cytokine-treated THP-1 cells. 44
Figure 12. Morphology of direct co-cultured THP-1, RWPE-1, and WPMY-1 cells. 45
Figure 13. Expression of M1 and M2 macrophage marker genes in directly co-cultured THP-1, RWPE-1, and WPMY-1 cells. 46
Figure 14. Establishment of indirect co-culture system of M2 macrophages and prostate cells. 48
Figure 15. Expression of fibrosis-related genes in prostate cells indirectly co-cultured with M2 macrophages. 49
Figure 16. Effects of 17β-estradiol and dihydrotestosterone on expression of fibroblast-related genes in 2D co-cultured RWPE-1 and WPMY-1 cells. 51
Figure 17. Expression of estrogen receptor and androgen receptor mRNA in THP-1 and M0, M1, and M2 macrophages. 53
Figure 18. Effects of 17β-estradiol and dihydrotestosterone on fibrosis in prostate cells indirectly co-cultured with M2 macrophages. 54
Figure 19. Effects of 17β-estradiol and dihydrotestosterone on the expression of fibroblast-related genes in the prostate cells indirectly co-cultured with M2 macrophages. 55
Figure 20. Representative immunoblot images of cytokines secreted by macrophages indirectly co-cultured prostate cells. 57
Figure 21. Cytokine profiles secreted by macrophages indirectly co-cultured with prostate cells after E2 and DHT treatment. 58