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I. 서론 8

II. 재료 및 실험방법 9

1. 재료 9

1) 실험동물 9

2) 약재 9

3) 세포 10

2. 실험방법 10

1) 약재추출 10

2) 화학분석방법 (LC/MS) 11

3) 골절 동물 모델 11

4) 동물실험 설계 12

5) in vivo 12

6) in vitro 15

3. 통계 분석 17

III. 결과 18

1. LC/MS 분석 18

2. in vivo 21

1) BMP2 유전자 발현에 미치는 영향 21

2) COX2 유전자 발현에 미치는 영향 23

3) Col2a1 유전자 발현에 미치는 영향 25

4) Sox9 유전자 발현에 미치는 영향 27

5) Runx2 유전자 발현에 미치는 영향 29

6) Osterix 유전자 발현에 미치는 영향 31

7) 뼈의 재형성에 미치는 영향 33

3. in vitro 35

1) 조골세포의 세포독성에 미치는 영향 35

2) 조골세포의 Osteocalcin, Runx2 유전자 발현에 미치는 영향 36

3) 대식세포의 세포독성에 미치는 영향 37

4) 대식세포의 TNF-α 발현에 미치는 영향 38

IV. 고찰 39

V. 결론 45

참고문헌 47

ABSTRACT 53

List of Tables

Table 1. The Herbal Composition of Danggwisu-san (DG) 10

Table 2. Nucleotide Sequence of Primer for Real-Time PCR 14

Table 3. Nucleotide Sequence of Primer for Real-Time PCR 16

Table 4. Summary of Metabolite Screening from DG. 20

List of Figures

Fig. 1. LC/MS Analysis of DG. 19

Fig. 2A, B. Effect of DG on BMP2 Expression in Bone Marrow from... 22

Fig. 3A, B. Effect of DG on COX2 Expression in Bone Marrow from... 24

Fig. 4A, B. Effect of DG on Col2a1 Expression in Bone Marrow from... 26

Fig. 5A, B. Effect of DG on Sox9 Expression in Bone Marrow from... 28

Fig. 6A, B. Effect of DG on Runx2 Expression in Bone Marrow from... 30

Fig. 7A, B. Effect of DG on osterix Expression in Bone Marrow from... 32

Fig. 8A-D. Effect of DG on Bone Regeneration in Femoral Shaft... 34

Fig. 9. Cytotoxic Effect of DG in MG63 cells. 35

Fig. 10A, B. Effect of DG on osteocalcin and Runx2 Expression in MG63... 36

Fig. 11. Cytotoxic Effect of DG in RAW264.7 cells. 37

Fig. 12. Effect of DG on TNF-α Production in RAW264.7 cells. 38

초록보기

Objective :

This study was designed to evaluate the effects of Danggwisu-san (Dangguixu-san, DG) on bone repair from femur fracture in mice.

Methods :

Mice were randomly divided into 4 groups (normal, control, positive control and DG 300 ㎎/㎏-treated group). Fractures were induced in the right femur using the 'Bonnarens and Einhorn' method except normal group. Mice in the normal group received no treatment at all. Those in the control group were orally administered with phosphate buffered saline whereas those in the positive control group were orally given tramadol (20 ㎎/㎏), once a day. Mice in the experimental group were orally medicated with DG once a day for 4 weeks.

In order to investigate the effects of DG on gene expressions in experimental animals with fracture, gene expression was evaluated on a phase basis after the injury. We measured the levels of BMP2 and COX2 genes expressed in bone marrow at the 3rd and the 7th day after the fracture (period referred to as inflammatory stage). We examined the levels of gene expressions in Sox9, Col2a1, Runx2, osterix genes in bone marrow at the 14th and the 28th day after the fracture (period referred to as soft callus stage, hard callus stage respectively) by real-time PCR. After the cytotoxicity test, we analyzed the levels of expression of osteocalcin and Runx2, the representative bone metabolic markers, and TNF-α, a pro-inflammatory cytokine. The process of fusion in the fracture was also investigated by gross examination

Results :

1. in vivo

1) Level of gene expression in BMP2 significantly decreased in the DG group at 3 days after the fracture and just increased 7th days after the fracture.

2) Level of gene expression in COX2 significantly decreased in the DG group at 3 days after the fracture and just increased at 7th days after the fracture.

3) Level of gene expression in Col2a1 increased in the DG group at 14th days after the fracture and decreased at 28th days after the fracture, but there was no significance.

4) Level of gene expression in Sox9 significantly increased in DG group at 14th days after the fracture and just decreased at 28th days after the fracture.

5) Level of gene expression in Runx2 increased in DG group at both 14th, 28th days after the fracture, but there was no significance.

6) Level of gene expression in osterix increased in DG group at 14th days after the fracture and decreased at 28th days after the fracture, but there was no significance.

7) The degree of unilateral fracture fusion investigated by gross examination was significantly faster than those of the other groups at the 7th, 14th and 28th day after fracture in the DG group respectively.

2. in vitro

1) Osteocalcin and Runx2 genes expressions increased when DG was treated in osteoblasts.

2) The level of TNF-α in macrophages was increased by DG in a dose-dependent mannerand and 250 and 500 ㎍/㎖ showed statistical significance.

Conclusions :

In conclusion, the results showed that after the occurrence of the fracture, DG promotes the healing of the fracture through the expression of bone repair-related genes and TNF-α production. We also investigated the process of fusion in the fracture by gross examination. This study may set the foundation for the clinical application of DG to the patients with bone fractures.

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