당질코르티코이드로 유도된 골다공증 모델에서 중간엽 줄기세포의 조골세포로의 분화에서 지실의 효과
The Fruit of Poncirius Trifoliata Promotes Mesenchymal Stem Cell Differentiation into Osteoblasts in Glucocorticoid-induced Osteoporosis Model
- 주제(키워드) 지실 , Poncirin , 당질코르티코이드 유도 골다공증
- 발행기관 아주대학교
- 지도교수 정윤석
- 발행년도 2011
- 학위수여년월 2011. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000011833
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
The fruit of Poncirus trifoliata is known to have effects on digestive system, cardiovascular system, and kidney function. The authors studied the effects of ethyl acetate extract of poncirus trifoliata on the activities of osteoblast and animal model. Effects of osteoblastic differentiation were measured by alkaline phosphatase (ALP) activity, osteopontin (OPN) protein expression and mineral nodule formation in dexamethasone (DEX)-treated MC3T3-E1 cells and primary bone marrow mesenchymal stem cells (BMSCs). Five fractions of ethyl acetate extract of poncirus trifoliata through column chromatography over a silica gel eluting in gradient system and combined based on their thin layer chromatography (TLC) pattern. Fourth subfraction (E4) of ethyl acetate extract was further purified by column chromatography over MCI gel eluting, then it was purified by repeated silica gel column chromatography to give naringin and poncirin, respectively. Bone mineral density (BMD) was measured before and after treatment with ethyl acetate extract, E4 and poncirin in glucocorticoid-induced osteoporotic (GIO) mice. In evaluation of bone microarchitecture, it was tested whether E4 and poncirin affects trabecular bone structure in GIO mice using micro-CT. The E4 and poncirin significantly increased trabecular bone structure parameters in GIO mice. A biomarker of bone-formation, bone-alkaline phosphatase (BALP), osteocalcin (OC), and a biomarker of bone-resorption, C-terminal telopeptides of type I collagen (CTX), were evaluated in the serum of the GIO mice. We surmised that if poncirin induce mesenchymal stem cell differentiation into osteoblast, it might contribute to increase bone mass. To address this issue, poncirin treated in C3H10T1/2 mesenchymal stem cell lines and primary BMSCs. Treatment of C3H10T1/2 cells and BMSCs with poncirin induced osteoblast differentiation through runt-related transcription factor2 (Runx2) expression and transcriptional activity, transcriptional coactivator with PDZ-binding motif (TAZ), OC expression and mineral nodule formation. Poncirin prevented adipocyte differentiation via inhibition of lipid droplet accumulation, PPAR-γ, and C/EBP-β expression. Also poncirin prevented osteoclast differentiation via inhibition of TRAP-positive MNCs, and regulation of OPG/RANKL ratio. During mesenchymal stem cell differentiation, DEX inhibited osteoblast differentiation through inhibition of ALP activity, Runx2, OC, osteoprotegerin (OPG) expression and mineral nodule formation. However, poncirin induced mesenchymal stem cell differentiation into osteoblasts in DEX-treated C3H10T1/2 cells. It was tested that whether DEX inhibited Runx2 expression through smurf1 expression in C3H10T1/2 cells. DEX induced Runx2 degradation through enhancement of smurf1 expression in the levels of mRNA and protein. DEX-induced Runx2 degradation was recovered by transfection of Smad ubiquitination regulatory factor1 (Smurf1) siRNA.
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TABLE OF CONTENTS
ABSTRACT i
TABLE OF CONTENTS iii
LIST OF FIGURES vi
Ⅰ. INTRODUCTION 1
A. Glucocorticoid-induced osteoporosis and Bone 1
B. The fruit of poncirus trifoliata 3
C. Mesenchymal stem cell (MSC) differentiation 5
D. Therapies for osteoporosis 7
E. Aims of study 8
Ⅱ. MATERIALS AND METHODS 10
A. MATERIALS 10
1. Animals and experimental procedures 10
2. Reagents 12
3. Plant material extraction and fractionation 12
B. METHODS 13
1. Cell culture 13
2. Osteoblast, osteoclast and adipocyte differentiation 14
3. Alkaline phosphatase (ALP) activity 15
4. Western blot analysis 15
5. Ubiquitination of Runx2 16
6. Isolation of RNA and RT-PCR, Real-time PCR 16
7. Nodule formation and mineralization 18
8. HPLC analysis 18
9. Oil Red O staining 19
10. Constructs, DNA transfection and reporter assay 19
11. Bone mineral density (BMD) measurement 20
12. Microfocal computed tomography (micro-CT) 20
13. Biochemical markers of bone turnover 21
14. Tartrate resistant acid phosphate (TRAP) staining 21
15. Statistical analysis 22
Ⅲ. RESULTS 23
A. Ethyl acetate extract of Poncirus trifoliata induced osteoblast differentiation
in vitro 23
B. Identifying components of ethyl acetate extract of Poncirus trifoliata
using HPLC 27
C. General observation and effects of GC pellet on bone loss in mice model 29
D. Ethyl acetate extract, E4 of Poncirus trifoliata and poncirin inhibited bone loss
in GIO model 31
E. E4 of Poncirus trifoliata and poncirin induced trabecular bone structural
properties in GIO model 33
F. Effects of E4 of Poncirus trifoliata and poncirin on biochemical markers
for bone turnover in GIO model 36
G. Effects of poncirin on mesenchymal stem cell (MSC) differentiation 38
H. Effects of poncirin on osteoclast differentiation in C3H10T1/2 cells and
primary bone marrow monocytes (BMMs) 44
I. Effects of poncirin on osteoblast and osteoclast differentiation in DEX-treated
C3H10T1/2 cells and BMSCs 46
J. DEX promoted Runx2 degradation through Smurf1 in vitro and poncirin
inhibited DEX-induced Smurf1 in vivo 49
Ⅳ. DISCUSSION 57
Ⅴ. CONCLUSION 62
REFERENCES 63
국문요약 72
LIST OF FIGURES
Fig. 1. The fruit of Poncirus trifoliata 4
Fig. 2. Multiple mechanisms of mesenchymal stem cells 6
Fig. 3. Animal experimental schedule for GIO 11
Fig. 4. Effect of DEX and ethyl acetate extract of Poncirus trifoliata on ALP activity 24
Fig. 5. Effect of ethyl acetate extract of Poncirus trifoliata on OPN protein expression
in MC3T3-E1 cells 25
Fig. 6. Alizarin Red S staining of mineralized bone nodules 26
Fig. 7. HPLC analysis 28
Fig. 8. Body weights and BMD analysis 30
Fig. 9. Effects of ethyl acetate extract of Poncirus trifoliata including E4 and poncirin
on BMD in GIO mice 32
Fig. 10. E4 of Poncirus trifoliata and poncirin improved trabecular bone
microarchitecture in GIO mice 34
Fig. 11. High resolution micro-CT images showing trabecular bone microarchitecture 35
Fig. 12. Serum levels of bone-ALP, OC and CTX in the GIO mice 37
Fig. 13. Poncirin inhibited adipocyte differentiation of the C3H10T1/2 cells and
primary BMSCs 40
Fig. 14. Poncirin promoted expression and transcriptional activity of Runx2 41
Fig. 15. Poncirin enhanced osteoblast differentiation in C3H10T1/2 cells 42
Fig. 16. Alizarin Red S staining of mineralized bone nodules 43
Fig. 17. Poncirin inhibited osteoclast differentiation 45
Fig. 18. Poncirin promoted osteoblast differentiation and inhibited osteoclast
differentiation 47
Fig. 19. DEX inhibited Runx2 expression in C3H10T1/2 cells 51
Fig. 20. DEX induced Runx2 degradation through GR in C3H10T1/2 cells 52
Fig. 21. DEX increased Smurf1 expression in vitro and in vivo 53
Fig 22. DEX induced Ubiquitination of Runx2 protein 54
Fig. 23. Smurf1 siRNA blocked DEX-induced Runx2 degradation 55
Fig. 24. Effect of poncirin on Smurf1 mRNA expression in GIO mice model 56
