sRANKL 클로닝 및 발현과 디아릴헵타노이드 허수테논의 파골세포생성에 대한 효과
Cloning and expression of sRANKL and effects of hirsutenone, diarylheptanoid, on osteoclastogenesis
- 주제(키워드) osteoclast , osteogenesis
- 발행기관 강릉원주대학교 일반대학원
- 지도교수 주성수
- 발행년도 2015
- 학위수여년월 2015. 2
- 학위명 석사
- 학과 및 전공 일반대학원 해양응용생명공학과
- 원문페이지 III, 54
- 실제URI http://www.dcollection.net/handler/kangnung/000000007019
- 본문언어 영어
초록/요약
Bone is continuously remodeled through new bone formation by osteoblasts and resorption of old bones by osteoclasts. The amounts of bone tissue are determined by the balance between the rates of bone formation and bone resorption during physiological growth and remodeling of skeleton. Among essential factors for osteoclastogenesis, receptor activator of NF-κB ligand (RANKL) has been focused because RANKL binds to the RANK receptor on osteoclast precursor and mature osteoclast cells which stimulate bone resorption. Thus, in this study, we primarily aimed to construct osteoclastogenesis system in RAW 264.7 cells by cloning soluble RANKL (sRANKL) and to examine the inhibitory effect of dearylheptanoid, hirsutenone, on osteoclastogenesis. Our results revealed that sRANKL was successfully cloned and expressed (approximately 35 kDa) in pET32a expression system and well induced osteoclastogenesis in Raw 264.7 cells. Moreover, osteoclast marker tartrate-resistant acid phosphatase (TRAP) and Osteoclast-associated receptor (OSCAR) were highly expressed when treated with cloned sRANKL in Raw 264.7 cells. The binding of sRANKL and RANK was clearly confirmed by immunohistochemistry (ICC). Meanwhile, hirsutenone effectively inhibited the expression of TRAP and OSCAR in sRANKL-treated Raw 264.7 cells and also inactivated cytosolic nuclear factor activated T-cell (NFATc), which can collaborate the bone resorption with macrophages. Taken together, we conclude that the cloned sRANKL successfully induced osteoclastogenesis in Raw 264.7 cell system within 5 days and hirsutenone would be the promising novel candidate for anti-oesteoporotic pharmacotherapies by suppressing osteoclastogenesis.
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ABSTRACT
LIST OF FIGURES
LIST OF TABLES
1. INTRODUCTION
2. MATERIALS AND METHODS
2.1 Reagents
2.2 Polymerase chain reaction (PCR)
2.3 Cloning of sRANKL gene
2.4 Analysis of gene sequences
2.5 Subcloning of sRANKL into pET32a expression vector
2.6 Overexpression of sRANKL protein
2.7 Western blot analysis
2.8 Amino acid analysis
2.9 Cell proliferation
2.10 Tartrate-resistant acid phosphatase (TRAP) – positive cell staining
2.11 CD4+ T lymphocyte from mice splenocytes
2.12 mRNA profiles of T-cell activation
2.13 Immunohistochemistry
2.14 Statistical analysis
3. RESULT
3.1 Gene cloning of sRANKL
3.2 Identification of cloned gene
3.3 Protein expression, purification and determination of sRANKL
3.4 Analysis of sRANKL amino acids
3.5 Osteoclastogenesis in Raw 264.7 cells
3.6 Immunohistochemistry analysis
3.7 Cytotoxicity of hirsutenone
3.8 Expression of TRAP in Raw 264.7 cells
3.9 Downstream signal pathways of sRANKL
3.10 Suppression of T lymphocyte activation by hirsutenone
4. DISCUSSION
REFERENCES
