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논문 기본 정보

자료유형
학위논문
저자정보

Jin Ju Park (부산대학교, 부산대학교 대학원)

지도교수
황대연
발행연도
2019
저작권
부산대학교 논문은 저작권에 의해 보호받습니다.

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이 논문의 연구 히스토리 (4)

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NKX3.1 is a well-known prostate tumor suppressor gene which acts as an androgen-regulated transcription factor. Functional defects of this gene lead to defects in the development of the prostate gland, including defects in prostatic ductal branching morphogenesis, secretions, and epithelial hyperplasia and dysplasia. However, knockout of NKX3.1 as a single gene using CRISPR/Cas9 has not been attempted. In this study, we investigate the effects of CRISPR/Cas9-mediated exon I deletion of NKX3.1 gene on the physiological phenotypes and prostate carcinogenesis. To achieve this, morphological, histological and biochemical alterations in the carcinogenesis of prostate as well as incidence of testosterone-induced prostate cancer were characterized in 16- and 24-week-old NKX3.1 knockout type (KO) mice bestowed the CRISPR/Cas9-mediated NKX3.1 mutant gene.
We first assessed the phenotypic characterization of C57BL/6N-NKX3.1em1Hlee KO mice with the CRISPR/Cas9-mediated NKX3.1 mutant gene at 16- and 24-week-old. The expression of NKX3.1 protein was significantly decreased in C57BL/6N-NKX3.1em1Hlee KO mice and heterozygous type (HT) mice compared to the wild type (WT) mice. However, no significant differences were observed with respect to genotype ratio, sex ratio, body weight, body temperature, body length, food intake, water consumption, survival rate, organs weight and blood analysis of WT, HT and KO mice for 16-week-old. Serum analysis revealed increased levels of only LDH in KO mice as compared to WT mice. The weight of dorsal-lateral prostate (DLP) and anterior prostate (AP) were enhanced in the C57BL/6N-NKX3.1em1Hlee KO mice at 24-week-old, while the ventral prostate (VP) weight was maintained at a constant level. Some significant decreases on the epithermal growth factors receptor (EGFR)-downstream signaling pathway, apoptotic proteins and anti-cancer proteins were obtained in the C57BL/6N-NKX3.1em1Hlee KO mice at 24-week-old. Furthermore, C57BL/6N-NKX3.1em1Hlee KO mice showed higher incidence for hyperplasia and adenoma in prostate.
In order to evaluate the incidence of testosterone-induced prostate cancer in the prostate of CRISPR/Cas9-mediated C57BL/6N-NKX3.1em1Hlee KO mice, we next examined alterations in the testosterone level, histopathological structure, metastasis related factor and apoptosis proteins in WT and KO mice after administering testosterone for 6 weeks. We observed that weights of only DLP and AP were significantly increased in testosterone-treated C57BL/6N-NKX3.1em1Hlee KO mice as compared to testosterone-treated WT mice, although the hormone levels were increased similarly. Also, the increase rate of hyperplasia and adenoma were greater in the testosterone-treated C57BL/6N-NKX3.1em1Hlee KO mice than those of control group. A similar pattern was detected for the expression of angiogenic proteins and apoptotic proteins in testosterone-treated C57BL/6N-NKX3.1em1Hlee KO mice. However, the dose-dependent decrease of the Bax/Bcl-2 ratio was observed in the C57BL/6N-NKX3.1em1Hlee WT mice.
Taken together, these results indicate that CRISPR/Cas9-mediated NKX3.1 mutant gene promotes the development of prostate cancer by enhancing DLP and AP weight, alteration of EGF receptor signaling pathway, and decreasing apoptotic proteins and cancer-related proteins. Also, our results show that testosterone may stimulate the incidence of prostate cancer by regulating the angiogenic and apoptotic proteins in C57BL/6N-NKX3.1em1Hlee WT mice. Furthermore, these results indicate that C57BL/6N-NKX3.1em1Hlee KO mice can be considered the most appropriate animal model for evaluating therapeutic effects and studying their action mechanism on the prostate cancer.

목차

I. INTRODUCTION 1
1. Prostate 1
1.1 Roles and structure of prostate 1
1.2 Development of prostate 4
1.3 Role of androgen receptor and testosterone in prostate 5
2. What is NKX3.1? 7
2.1 NKX3.1 transcription factor 7
2.2 Structure properties of the NKX3.1 gene and protein 8
3. Production and phenotypes of NKX3.1 KO mice 15
3.1. Production strategy for NKX3.1 KO mice 15
3.2. Phenotypic features of NKX3.1 KO mice 17
II. MATERIALS AND METHODS 24
1. Phenotypical characterization of C57BL/6N-NKX3.1em1Hlee KO mice 24
1.1 Production and identification 24
1.2 Animals care and use 26
1.3 Analysis for litter size, sex composition and genotype 26
1.4 Measurement of body and organs weight 28
1.5 Measurement of food intake and water consumption 28
1.6 Analysis for survival rate and clinical sign 28
1.7 Whole blood and serum analysis 29
1.8 Measurement of body temperature and body length 30
1.9 Histological analysis 30
1.10 Immunohistochemical analysis (IHC) 31
1.11 Immunofluorescence analysis (IF) 31
1.12 Western blot 32
1.13 Enzyme-linked immunosorbent assay (ELISA) 33
1.14 Statistical analysis 33
2. Incidence of testosterone-induced prostate cancer in the CRISPR/Cas9-mediated C57BL/6-NKX3.1em1Hlee KO mice 34
2.1 Experimental design 34
2.2 Western blot 35
III. RESULTS 36
1. Phenotypical characterization of C57BL/6N-NKX3.1em1Hlee KO mice 36
1.1 Effects of CRISPR/Cas9-mediated NKX3.1 deletion on the genotype and sex ratio 36
1.2 Effects of CRISPR/Cas9-mediated NKX3.1 deletion on the survival rate until 16-week-old 36
1.3 Effects of CRISPR/Cas9-mediated NKX3.1 deletion on body temperature, body length and organs weight in 16-week-old 39
1.4 Effects of CRISPR/Cas9-mediated NKX3.1 deletion on weight gain and feeding behavior until 16-week-old 39
1.5 Effects of CRISPR/Cas9-mediated NKX3.1 deletion on blood and serum components in 16-week-old 45
1.6 Effects of CRISPR/Cas9-mediated NKX3.1 deletion on weights in prostate 45
1.7 Effects of CRISPR/Cas9-mediated NKX3.1 deletion on confirmation ofhistological difference 50
1.8 Effects of CRISPR/Cas9-mediated NKX3.1 deletion on EGFR downstream signaling pathway 59
1.9 Effects of CRISPR/Cas9-mediated NKX3.1 deletion on apoptotic response and tumor related genes 59
2. Incidence of testosterone-induced prostate cancer in the CRISPR/Cas9-mediated C57BL/6-NKX3.1em1Hlee KO mice 73
2.1 Effects of testosterone injection on testosterone level in serum 73
2.2 Effects of testosterone injection on alteration in the body weight and feeding behavior 73
2.3 Effects of testosterone injection on conformation and weights of prostate 78
2.4 Effects of testosterone injection on histopathology of prostate each lobe 78
2.5 Effects of testosterone injection on expression of proteins in prostate 81
IV. DISCUSSION 88
1. Phenotypical characterization of C57BL/6N-NKX3.1em1Hlee KO mice 88
2. Incidence of testosterone-induced prostate cancer in the CRISPR/Cas9-mediated C57BL/6N-NKX3.1em1Hlee KO mice 91
V. REFERENCES 98
SUMMARY (In Korean) 110
Curriculum Vitae 113

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