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The Role of NFIC and CPNE7 in Regulation of Cartilage Development and Dentin Regeneration : 연골 발달과 상아질 재생에서 NFIC와 CPNE7의 역할

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dc.contributor.advisor박주철-
dc.contributor.author노송이-
dc.date.accessioned2024-05-31T18:02:36Z-
dc.date.available2024-05-31T18:02:36Z-
dc.date.issued2022-
dc.identifier.other000000169794-
dc.identifier.urihttps://hdl.handle.net/10371/204057-
dc.identifier.urihttps://dcollection.snu.ac.kr/common/orgView/000000169794ko_KR
dc.description학위논문(박사) -- 서울대학교대학원 : 치과대학 치의과학과, 2022. 2. 박주철.-
dc.description.abstractGrowth plate chondrocytes are organized in parallel columns comprising resting, proliferative, pre-hypertrophic, and hypertrophic zones. A specialized cartilage matrix is deposited as chondrocytes mature and serves as a scaffold for blood vessels and osteoblasts to invade, leading to bone matrix accumulation (Arsenault et al. 1988; Hunziker et al. 1999; Noonan et al. 1998). Disruption in this multi-step differentiation process could lead to skeletal anomalies such as dwarfism (LuValle and Beier 2000).
Herein, the essential role of nuclear factor I C (NFIC) in epiphyseal cartilage formation was demonstrated by examining the femoral growth-plate of Nfic-deficient mice. Chondrocyte proliferation was downregulated, and the number of apoptotic cell was increased in the growth plates of Nfic-/- mice. Further, the expression of the cell cycle inhibitor p21 was upregulated in the primary chondrocytes of Nfic-/- mice, whereas that of cyclin D1 was downregulated. These results suggest that NFIC may contribute to postnatal chondrocyte proliferation by inhibiting p21 expression and by increasing the stability of cyclin D1 protein.
During dentinogenesis, neural-crest derived ectomesenchymal stem cells differentiate into odontoblasts via sequential and reciprocal interaction with dental epithelium. Odontoblasts are polarized, highly specialized post-mitotic cells that are responsible for the dentin secretion. Besides their post-mitotic nature, the complex regulatory mechanism required for odontoblast differentiation has been considered as a big hurdle for tooth regeneration.
Copine 7 (CPNE7), a dental epithelium-derived factor, can re-activate dormant odontoblasts, and promote tubular tertiary dentin formation. In the present thesis, CPNE7 was suggested to promote tubular tertiary dentin formation by fine-tuning canonical Wnt signaling activity via modulation of β-catenin protein stability. Furthermore, the observed defects in primary ciliogenesis and altered expression of cell cycle regulators suggests a role for CPNE7 in cell cycle regulation during the terminal differentiation of odontoblasts.
Taken together, above studies suggest the essential roles of NFIC and CPNE7 during the epiphyseal chondrocyte proliferation and terminal odontoblast differentiation during dentin regeneration, respectively.
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dc.description.abstract중간엽 줄기세포의 응집 (condensation)과 분화를 통해 만들어진 골단의 성장판 연골세포 (epiphyseal growth plate chondrocyte)는 장골 (long bone)의 지속적인 길이 성장을 담당한다. 본 연구에서는 nuclear factor I C (Nfic)의 결핍이 넓적다리 뼈의 성장판 형성에 미치는 영향에 대해 밝혔다. Nfic가 결핍된 쥐의 성장판에서 연골세포의 증식이 감소하고 사멸세포 (apoptotic cell)의 수가 증가했으며, 세포 주기 억제 인자인 p21 발현의 증가와 cyclin D1 발현의 감소가 관찰되었다. 본 연구를 통해 NFIC가 p21을 억제하고 cyclin D1 단백질의 안정성을 증가시킴으로써 생후 연골세포의 증식에 관여할 가능성이 있음을 밝혔다.
치아 형성 과정동안 상아모세포(odontoblast)는 신경능선세포 기원의 외배엽성 미분화중간엽세포 (ectomesenchymal stem cell)로부터 분화한다. 유사핵분열중지(postmitotic) 세포인 상아모세포로의 완전한 분화는 치성 상피와 간엽 간의 순차적인 상피간엽상호작용(epithelial mesenchymal interaction, EMI)의 정밀한 조절을 필요로 하는데, 아직까지는 그 조절 기전의 재현을 통한 생리적 상아질 재생이 불가능하기 때문에 관련 연구가 계속해서 이뤄지고 있다. 치성 상피에서 유래한 인자인 copine 7 (CPNE7)은 생체 내에서 삼차 상아질 형성을 유도하고, 생체 외 배양 실험에서 상아모세포의 분화와 석회화를 유도한다고 보고되어 있다. 본 연구에서는 CPNE7이 β-catenin 단백질의 안정성 조절을 통해 Wnt 신호전달 체계의 활성을 조절함으로써 상아세관을 가진 삼차상아질의 형성에 기여할 가능성이 있음을 밝혔다. CPNE7에 의한 Wnt 신호전달 체계 활성 조절은 세포주기의 조절과도 연관이 있는데, 분화 과정 동안 상아모세포의 유사핵분열중지 상태를 유지시킴으로써 최종 분화(terminal differentiation)를 유도할 가능성에 대해서도 보고했다.
총괄적으로, 위의 연구들은 NFIC와 CPNE7이 성장판 연골모세포의 증식 및 분화 과정과, 중간엽 줄기세포로부터 상아모세포가 분화하는 과정에서 각각 중요한 역할을 할 가능성을 시사한다.
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dc.description.tableofcontentsAbstract 4
Table of Contents 7
List of Figures 11
Abbreviations 13
Chapter 1. Literature Review 16
I. Cell cycle control during endochondral ossification 16
II. Nuclear factor I C in hard tissue formation 17
III. Tubular dentin formation 17
IV. Wnt/β-catenin signaling pathway in dentinogenesis 18
V. Copine 7 in dentin regneration 19
VI. Rationale and outline of the thesis experiments 20
Chapter 2. NFIC is Required for Epiphyseal Chondrocyte Proliferation during Postnatal Cartilage Development 21
I. Introduction 22
II. Materials and Methods 24
1. Mice 24
2. Microcomputed tomography (micro-CT) analysis, histology, and immunohistochemistry 24
3. Cell culture and transfection 25
4. MTT assays 26
5. Terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling (TUNEL) and DNA fragmentation assays 26
6. Reverse transcription-polymerase chain reaction (RT-PCR) and real time PCR analysis 27
7. Western blot analysis 28
8. Statistical analysis 29
III. Results 30
1. Decreased femoral growth-plate length in Nfic-deficient mice 30
2. Downregulated chondrocyte proliferation in the femoral growth plate of Nfic-deficient mice 35
3. Increased chondrocyte apoptosis in the femoral growth plates of Nfic-deficient mice 39
4. NFIC regulates the stability of cyclin D1 protein 41
5. Upregulated MMP9 and MMP13 levels in Nfic-/- mice chondroctyes 45
IV. Discussion 47
Chapter 3. CPNE7 Regulates Canonical Wnt Signaling Activity via Modulation of β-catenin Stability during Tertiary Dentin Formation 53
I. Introduction 54
II. Materials and Methods 58
1. Mice 58
2. Cell cuture and transfection 58
3. Western blot analysis 59
4. Real-time PCR 60
5. Alizarin red S staining 62
6. Immunofluorescence 62
7. Statistical analysis 63
III. Results 64
1. Sustained β-catenin expression during differentiation of Cpne7-deficient mDPCs 64
2. Defective mineralization in differentiating Cpne7 -/- mDPCs 68
3. CPNE7 induces proteasomal degradation of β-catenin in MDPC-23 cells 71
4. Stage specific inhibition of tankyrase activity partially rescued mineralization in Cpne7 -/- mDPCs 73
5. Cpne7 deficiency led to altered expression of cell cycle regulators and defective primary ciliogenesis 76
IV. Discussion 79
Chapter 4. Concluding Remarks 84
References 86
Abstract in Korean 96
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dc.format.extentxii, 82-
dc.language.isoeng-
dc.publisher서울대학교 대학원-
dc.subjectNfic-
dc.subjectCpne7-
dc.subjectproliferation-
dc.subjectdifferentiation-
dc.subjectregeneration-
dc.subjectodontoblasts-
dc.subjectchondrocytes-
dc.subject.ddc617.6-
dc.titleThe Role of NFIC and CPNE7 in Regulation of Cartilage Development and Dentin Regeneration-
dc.title.alternative연골 발달과 상아질 재생에서 NFIC와 CPNE7의 역할-
dc.typeThesis-
dc.typeDissertation-
dc.contributor.AlternativeAuthorSong Yi Roh-
dc.contributor.department치과대학 치의과학과-
dc.description.degree박사-
dc.date.awarded2022-02-
dc.contributor.major세포 및 발생생물학-
dc.identifier.uciI804:11032-000000169794-
dc.identifier.holdings000000000047▲000000000054▲000000169794▲-
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