학위논문(박사)--서울대학교 대학원 :의과대학 의학과,2019. 8. 이재협.Osteoporosis is caused by an imbalance between bone formation and bone resorption that results in low bone mass and deteriorated bone microstructure and finally elevates the risk of low-trauma fracture. For developing new therapies for managing osteoporosis, this study compromised 3 stages as follows: 1, the bone formation efficacy of recombinant human bone morphogenetic protein 2 (rhBMP-2) was investigated since bone substitute is necessary for osteoporosis patients; 2, the efficacy of currently available medications was analyzed via meta-analysis; 3, the impact brought by the mutation in Drg2 in bone homeostasis was researched. Their methods and results were as follow. In the first part, we compared the osteoinductivity of Escherichia coli rhBMP-2 (ErhBMP-2) with Chinese hamster ovary cell-derived rhBMP-2 (CrhBMP-2) with human mesenchymal stem cells and rat calvarial defect. In the second part, we systematically reviewed the effect of current osteoporosis medications on preventing secondary osteoporotic vertebral and non-vertebral fractures from randomized controlled studies and synthesized their result via meta-analysis. In the third part, we compared the transcription level of DRG2 in osteoporosis and non-osteoporosis subjects, and furtherly fabricated Drg2 knockout mice and analyzed the difference in bone phenotype of wild type and Drg2 knockout mice. At the end of this part, we investigated the possible mechanisms and signals Drg2 involved in osteoblastic differentiation. The results from the first part showed ErhBMP-2 could have comparable osteoinductivity with Chinese hamster ovary cell-derived BMP-2 while using the demineralized bone matrix as the carrier. In the second part, we found the medications could have a consistent effect on osteoporosis patients, regardless of their fracture history. And in the third part, we found osteoporosis patients had higher expression level of Drg2 and knocking out of Drg2 in mice significantly improved bone mass and mineral density even if mice were ovariectomized. The bone marrow-derived macrophage in Drg2 knockout mice showed lower osteoclastogenesis while the bone marrow mesenchymal stem cell concurrently showed higher osteoblastogenesis than wild type mice. Furtherly, inhibition of Drg2 expression in mouse MC3T3-E1 cells elevated its osteogenicity via canonical and non-canonical BMP pathway. In summary, we found the ErhBMP-2 might have the potential of being used as an anabolic agent for osteoporosis fracture; currently available medications could have a significant effect on preventing secondary osteoporotic fracture; and Drg2 as an important regulator in bone remodeling, which suggested Drg2 inhibitor could be a potential anabolic for treating osteoporosis.골다공증은 골량 감소와 골 미세구조 이상을 야기하는 질환으로 골형성과 골흡수간 불균형에 의해 발생하며, 저손상 골절의 위험을 증가시키는 질환이다. 새로운 골다공증 치료법을 개발하기 위하여 다음 3단계의 연구를 진행하였다. 골다공증 환자는 골대체제가 필요하기 때문에 재조합 골형성단백질 제2형(rhBMP-2) 의 골형성 효능 연구, 현재 사용되는 골다공증 치료제의 2차 골절예방 효능에 관한 메타 분석 연구와 함께, developmentally regulated GTP binding protein 2 (Drg2) 의 골 항상성에 미치는 영향을 연구하였다. 첫번째 연구에서는, 대장균 유래 골형성 단백질 제2형(ErhBMP-2)과 동물세포 유래 골 형성 단백질 제2형(CrhBMP-2)의 골유도성을 인간 간엽줄기세포 및 랫드 두개골 결손모델에서 비교하였고 두번째 연구에서는, 기존 골다공증 치료제가 골다공증성 척추 및 비척추 골절을 예방하는 효과에 대하여 메타분석을 시행하였으며 세번째 연구에서는, 골다공증이 있는 환자군과 정상 대조군의 골수 유래 간엽줄기세포 에서 DRG2의 발현을 비교하였고, Drg2 결손 마우스를 제작하여 대조군과 골 표현형의 차이를 분석하였다. 또한 Drg2 가 조골세포의 분화에 관여하는 기전과 신호전달 연구를 수행하였다. 첫번째 연구에서 ErhBMP-2가 탈회골기질을 담체로 사용할 때 CrhBMP-2와 유사한 골 유도능력을 가질 수도 있음을 확인하였으며 두번째 연구에서는 골다공증 환자의 골절 병력과 무관하게 약물치료가 지속적인 영향이 있을 수 있다는 것을 알 수 있었습니다. 세번째 연구에서 골다공증 환자는 Drg2 mRNA 발현 정도가 정상 대조군보다 더 높았고, Drg2 결손마우스는 난소절제술을 시행하였을 때 골량 보호 효과가 관찰되었다. Drg2 결손 마우스에서 얻은 골수유래 대식세포를 대조군과 비교했을 때, 파골세포 분화력이 낮았으며 골수유래 줄기세포의 조골세포 분화력은 높았다. 또한 마우스 MC3T3-E1 세포에서의 Drg2 발현을 억제시키면 정식 및 비정식 BMP 경로를 통하여 조골세포의 분화가 증가하였다. 결론적으로, 본 연구에서는 ErhBMP-2가 골다공증성 골절에서 동화제제로서 사용 될 수 있는 가능성과 Drg2 유전자가 골 재형성에 있어 중요한 조절 인자임을 확인하였다.Abstract I
Table of Contents: IV
List of Tables VI
List of Figures VII
Introduction 1
Methods 5
Osteoinductive treatment of human mesenchymal stem cells 5
ALP staining and and ALP activity assay 5
Calcium staining and assay 6
Real-time PCR 7
Rat calvarial defect model 7
Micro-CT evaluation 8
Hematoxylin and eosin staining 9
Search for studies 10
Selection of studies 10
Data extraction and risk of bias 11
Data analysis and quality of evidence 12
Extraction of mesenchymal stem cells from human 13
Fabrication of DRG2 knock out mouse 13
Genomic typing and gender determination 14
Primary culture of BMMCs and bone marrow MSC 14
TRAP staining 15
shRNA transfection of MC3T3-E1 cell 15
Inducing osteoblastic differentiation in MC3T3-E1 cells 16
Western blot 16
Semiquantitative RT-PCR 17
Feeding and maintaining 17
Serum P1NP and CTX measurement 18
Ovariectomy 18
Calcein labeling 19
Statistics 19
Results 20
ALP assay 20
ALP staining 20
Calcium assay 20
Alizarin red staining 21
Real-time PCR 21
Animal experiments 21
Characteristics of included studies and risk of bias 22
Comparison with control group 24
Comparison between interventions 29
Higher DRG2 expression correlates with lower BMD 30
Knocking out of Drg2 affects mice postnatal bone formation 30
Inhibition of DRG2 improves bone architecture and BMD even in ovariectomized mice 31
Results of the GO enrichment and KEGG pathway analysis 33
Inhibiting the expression of DRG2 inhibits the osteoclastic differentiation of BMMCs and elevates osteoblastic differentiation of bone marrow MSCs 33
Inhibiting the expression of DRG2 elevates osteogenicity of MC3T3-E1 cells 34
Inhibition of DRG2 elevates OB differentiation via canonical and non-canonical BMP signaling 35
Discussion 37
The first section 38
The second section 41
The third section 46
References 50
Figures 61
Table 90
Supplementary material 104
논 문 초 록 123
Acknowledgments 126Docto
