골다공증에서의 Annexin A6의 역할
The Role of Annexin A6 in Osteoporosis
- 주제(키워드) osteoporosis , Annexin A6 (ANXA6) , osteoblast , bone mineral density (BMD) , single nucleotide polymorphism (SNP)
- 발행기관 아주대학교
- 지도교수 정선용
- 발행년도 2011
- 학위수여년월 2011. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000011786
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Osteoporosis is a common calcium and metabolic skeletal disease which is characterized by decreased bone mass, microarchitectural deterioration of bone tissue and impaired bone strength, thereby leading to enhanced risk of bone fragility. In this study, we aimed to discover a novel herbal therapeutic drug for effective osteoporosis treatment and to further clarify its molecular mechanism of action. At first, ethanol or methanol extracts of 68 edible Korean native plants were screened and Poncirus trifoliata (PT) was selected as a final candidate because of its high inhibitory activity on glucocorticoid (GC)-induced apoptosis plus its novelty. The hexane extract of PT (PT-H) inhibited apoptotic cell death of osteoblastic cell lines, C3H10T1/2 and MC3T3-E1 induced by synthetic GC, dexamethasone (Dex). In vivo mouse results indicated that PT-H not only had an inhibitory effect on the bone loss caused by GC, but also promoted bone formation. We further clarified the molecular mechanisms behind the effect of PT-H on GC-induced osteoporosis (GIO) by screening of differentially-expressed genes (DEGs) between Dex-induced osteoblastic cells with or without PT-H treatment. Finally, we found that the expression level of AnxA6 in Dex-induced osteoblastic cells and prednisolone-treated GIO-model mice was significantly decreased by PT-H treatment. These findings suggest that PT-H has a strong in vitro and in vivo inhibitory effect on GIO, and decreased expression of AnxA6 may play a key role in this inhibition. As the next step, we aimed to determine whether ANXA6 gene was associated with the susceptibility to osteoporosis and further identify novel genes for susceptibility to osteoporosis. At first, we performed a whole-genome comparative expression analysis. Ten genes were identified as the DEGs in the Dex-treated MC3T3-E1 cells. To validate and evaluate the identified DEGs, we performed quantitative real-time RT-PCR using the gene-specific primers and compared the expression levels of these genes between the Dex-treated and untreated MC3T3-E1 cells, and between the ovariectomized (OVX) and sham mice and found that the expression levels of 7 genes, Anxa6, Col5a1, Col6a2, Eno1, Myof, Nfib and Scara5, were significantly altered in the in vitro and in vivo osteoporosis models. Lastly, to determine whether the genetic variations of these 7 genes were associated with bone density and osteoporosis phenotypes in humans, we performed the quantitative bone density analysis and osteoporosis case-control analysis of 116 ingle nucleotide polymorphism (SNPs) in these 7 genes in the Korean Women’s Cohort (3570 subjects). There was a significant association between the SNPs in the 5 genes, ANXA6, COL5A1, ENO1, MYOF and SCARA5, and bone density and/or osteoporosis. Especially, the SNPs in the ANXA6 gene showed a highly significant association with bone density and their p-values satisfied the Bonferroni-corrected significance level. These results indicate that genetic variation in the ANXA6 gene is significantly associated with bone density and osteoporosis. This study may provide insight into the genetic basis of osteoporosis.
more목차
ABSTRACTⅰ
TABLE OF CONTENTS iii
LIST OF FIGURES vi
LIST OF TABLES viii
Ⅰ. INTRODUCTION 1
A. In vitro and in vivo inhibition of glucocorticoid-induced osteoporosis by the
hexane extract of Poncirus trifoliata 1
B. Genetic association between single nucleotide polymorphisms in the ANXA6 gene
and bone density/osteoporosis 3
Ⅱ. MATERIALS AND METHODS 6
1. Chemicals 6
2. Plant materials and preparation of hexane extracts 6
3. Cell culture 7
4. Cell viability assay 8
5. Terminal deoxynucleotidyl Transferase (TdT)-mediated dUTP nick end
labeling (TUNEL) staining 8
6. GIO mouse model experiment 9
7. Measurement of bone mineral density for density 10
8. Tissue sample preparation 10
iv
9. Annealing control primer (ACP)-based differential display reverse-transcriptase
polymerase chain reaction (RT-PCR) 10
10. Cloning and sequence analysis 12
11. Gene-specific quantitative real-time RT-PCR 12
12. Protein extraction and western blot analysis 14
13. Ovariectomized (OVX) mouse model 15
14. Human subjects 15
15. Genotyping and selection of SNPs 17
16. Statistical analysis 17
Ⅲ. RESULTS 19
A. In vitro and in vivo inhibition of glucocorticoid-induced osteoporosis by the
hexane extract of Poncirus trifoliata 19
1. Screening of the novel, effective natural sources on GIO 19
2. In vitro inhibition of PT-H on Dex-induced apoptosis 23
3. Inhibitory effect of PT-H on an in vivo GIO model 26
4. Screening and identification of DEGs in Dex-induced osteoblastic cells with
and without PT-H treatment 28
5. Validation of the identified DEGs by real-time RT-PCR and western blot
analysis 31
6. Effect of PT-H on the expression of AnxA6 in GIO-mice 34
B. Genetic association between single nucleotide polymorphisms in the ANXA6
gene and bone density/osteoporosis 36
v
1. Study design 36
2. Screening and identification of the DEGs in Dex-treated osteoblastic cell line
model 38
3. Validation of the identified DEGs in the cell line model by quantitative
real-time RT-PCR 43
4. Evaluation of the identified DEGs in the ovariectomized mouse model by
quantitative real-time RT-PCR 45
5. Association analysis of the genetic variation in the identified DEGs with bone
density and osteoporosis in humans 47
Ⅳ. DISCUSSION 60
A. In vitro and in vivo inhibition of glucocorticoid-induced osteoporosis by the
hexane extract of Poncirus trifoliata 60
B. Genetic association between single nucleotide polymorphisms in the ANXA6
gene and bone density/osteoporosis 65
Ⅴ. CONCLUSION 71
REFERENCES 72
국문요약 85
