골수 유래 중간엽 줄기세포의 연골화 분화에 저강도 초음파의 자극이 미치는 영향
Effects of Low Intensity Ultrasound on the in vitro Chondrogenic Differentiation of Mesenchymal Stem Cells Derived from Bone Marrow
- 주제(키워드) Low-intensity ultrasound; chondrogenic differentiation; bone marrow mesenchymal stem cells; alginate culture; viability; apoptosis; surface markers; flow cytometry
- 주제(KDC) 570
- 주제(DDC) 660
- 발행기관 아주대학교
- 지도교수 민병현
- 발행년도 2007
- 학위수여년월 2007. 8
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 본문언어 영어
초록/요약
Bone marrow mesenchymal stem cells (MSC) are known as progenitor cells capable of differentiation into osteoblasts, chondrocytes, adipocytes, myocytes. Many growth factors, cytokines and mechanical stimulation are known to affect chondrogenic differentiation of MSC, however, the mechanisms of how these factors affect chondrogenic differentiation remain unclear. To date, three-dimensional (3-D) culture and TGF-β are known as one of essential factors for chondrogenic differentiation. Recently, low-intensity ultrasound (LIUS) has been demonstrated to have the effects on the enhancement of fracture healing via increasing the chondrogenesis, which suggests that LIUS could affect in chondrogenic differentiation of MSC. The aim of these studies is to investigate the effect of LIUS treatment on the chondrogenic differentiation of MSCs. When LIUS is applied on the chondrogenesis of rabbit MSCs (rMSCs) in a 3-D alginate culture and after replating them on a monolayer culture, we found the increases of the matrix formation, the expression of chondrogenic markers such as collagen type II, aggrecan, and Sox-9, the expression of tissue inhibitor of metalloprotease - 2 implicated in the integrity of cartilage matrix, and the capacity to maintain the chondrogenic phenotypes in a monolayer culture. The 3-D alginate culture and the TGF-β1 treatment on chondrogenic differentiation system of human MSCs (hMSCs) resulted in the decrease of cell viability, which appeared to be mediated by apoptosis. In contrast, co-treatment of LIUS clearly enhanced cell viability and inhibited apoptosis under the same condition. The LIUS effect on the apoptotic event was further supported by the changes in the expression of apoptosis/viability related genes of p53, bax, bcl-2 and PCNA. Next, we examined the surface markers showing specific changes during the chondrogenic differentiation and dedifferentiation of hMSCs. hMSCs from adult bone marrow were subjected to chondrogenic differentiation in 3-D alginate culture with TGF-β3 for 2 weeks, followed by dedifferentiation in monolayer for 1 week. Surface antigens were selected from those showing expression changes during dedifferentiation of human articular chondrocytes (hACs). The results from flow cytometry identified 3 groups of surface antigens with differential expression patterns, but showed quite different from those shown in hACs reported previously. The groups 1 and 2 antigens were expressed at high levels in hMSCs. The expression of group 1 antigens (CD44, CD58, CD90, CD105 and CD166) decreased non-specifically just by 3-D alginate culture, while that of group 2 antigens (CD49c, CD49e, CD81 and CD151) decreased depending on the chondrogenic differentiation. The expression of group 3 antigens was not significantly observed at all experimental stages. Interestingly, the LIUS treatment showed no effect at all on the changes in their expression during the stages. Notably, LIUS effects were clearly shown even without TGF-β treatment. These finding demonstrate that LIUS stimulated chondrogenic differentiation of bone marrow MSC. LIUS treatment could be a valuable tool for cartilage tissue engineering using MSCs by enhancing the cell viability as well as by directing the chondrogenic differentiation process, its well-known activity. Our observation shows that the noninvasive ultrasound function as a kind of chondrogenic mechanical stimulus suggests potential clinical applications in cartilage repair.
more목차
Dessertation approval i
Table of Contents ii
List of Figures vi
List of Tables ix
ABSTRACT x
INTRODUCTION 1
1. Mesenchymal stem cells 1
2. Chondrogenic differentiation microenvironment 4
3. Low intensity ultrasound (LIUS) 7
Chapter I.
Low intensity ultrasound stimulation enhances the chondrogenic differentiation in alginate culture of mesenchymal stem cells 14
A. Materials and Methods 15
1. Isolation and culture of bone marrow mesenchymal stem cells 15
2. Alginate culture 15
3. Treatment of ultrasound and TGF-β3 16
4. Analysis of chondrogenic phenotypes in a monolayer culture 19
5. RT-PCR 19
6. Western blot analysis 21
7. Histological and immunohistochemical evaluation of alginate cultures 21
8. Statistical analysis 22
B. Result 23
C. Discussion 35
Chapter II.
Low intensity ultrasound inhibits apoptosis and enhances viability of human mesenchymal stem cells in three-dimensional alginate culture during chondrogenic differentiation 40
A. Materials and Methods 41
1. Isolation and culture of human bone marrow MSCs 41
2. Chondrogenic differentiation of hMSCs in alginate layer culture 42
3. Treatment of ultrasound and TGF-β1 42
4. Examination of cell viability 43
5. Lactate dehydrogenase (LDH) activity assay 43
6. Live/Dead cytotoxicity assay 43
7. TUNEL assay 44
8. Bax/bcl-2 expression by immunohistochemistry 44
9. Western blot analysis 45
10. RT-PCR 46
11. Statistical analysis 46
B. Result 47
1. LIUS treatment enhanced viability of hMSCs during chondrogenic differentiation 47
2. The LIUS treatment inhibited apoptosis of hMSCs during the chondrogenesis 50
3. The LIUS treatment inhibited the expression of apoptosis related genes 53
4. LIUS treatment enhanced the chondrogenic differentiation of hMSCs 53
5. LIUS treatment inhibited apoptotic event on the induced apoptosis of hMSCs by staurosporine 57
C. Discussion 63
Chapter III.
Changes in surface markers of human mesenchymal stem cells during the chondrogenic differentiation and dedifferentiation processes in vitro 66
A. Materials and Methods 67
1. Isolation and culture of human bone marrow MSCs 67
2. Chondrogenic differentiation of hMSCs in alginate layer culture 68
3. Dedifferentiation of differentiated cells in a monolayer culture 69
4. RT-PCR 69
5. Flow cytometry 70
B. Result 72
1. Chondrogenic differentiaton and dedifferentiation of hMSCs 72
2. Effect of TGF-β3 treatment on the expression of selected set of surface antigens 75
3. Effect of LIUS on the expression of typical MSCs markers 82
4. Early changes in the expression of group 1 surface antigens 84
C. Discussion 86
CONCLUSION 91
REFERENCE 92
국문요약 106
