MC3T3-E1 조골세포에서 가자 추출물에 의한 골형성능 분석

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Osteogenic effect of Terminalia cebula Retzius extract in pre-osteoblastic cell, MC3T3-E1

Sang-Yoen Park
Advisor : Prof. Su-Gwan Kim, DDS, Ph.D.
Department of Dental Biology
Graduates School of Chosun University

The World Health Organization (WHO) has advised that the use of natural products and plant extracts be investigated and a number of clinical studies have shown the effects of bone formation prepared from extracts of herbs. The plant Terminalia chebula (family Combretaceae, T. chebula) has been reported to possess antimicrobial and anticariogenic properties in addition to its antidiabetic, hepatoprotective, anti-inflammatory, antioxidant and many other effects. The ripe fruits of T. chebula have been used in the prevention and treatment of dental caries, gingivitis and stomatitis and previous studies demonstrated that T. chebula containing mouth rinses have antimicrobial effects against oral bacteria. The plant has a long history of use and is considered safe. As the increasing of elderly population due to development of clinical health care increases and the lifestyle of westernized eating, patients with requirement of bone graft in the periodental disease have being rapidly increased in the modern society. Therefore, the screening of medicinal herbal plants associated with bone formation have being required to develop the therapeutic clinical reagent in the dental science.

In the present study, T. chebula Retzius extracts have been shown the similar metabolic features that appear in the process of bone formation in vivo and osteoblast cell proliferation, differentiation, and calcification in mouse calvaria-derived MC3T3-E1 osteoblast cells. The detailed results are as follows.

1. To access the cell cytotoxicity of T. chebula, both human normal oral keratinocytes and MC3T3-E1 mouse osteoblastic cells were stimulated with 10, 50 and 100 µg/mL of T. chebula methanol extract for 24 hrs. After stimulation, the cell cytotoxicity on the both cells were accessed by MTT assay and Cell live & Dead assay. The methanol extract of T. chebula did not show the cell cytotoxicity in both cells. Furthermore, cell linve & dead assay have been showed the similar results with MTT assay.

2. The activities and mRNA level of alkaline phosphatase were accessed by Alkaline phosphatase activity assay and quantitative PCR, respectively. The alkaline phsophatase activity was significantly up-regulated by T. chebula in the MC3T3-E1 mouse osteoblastic cells as the dose dependent manner. Furthermore, the mRNA level of alkaline phsophatse was increased by T. chebula as well as the result of alkaline phosphatase activity, dose-dependently. To evaluate day-course of ALP activity of T. chebula extracts.

3. The mineralization of MC3T3-E1 mouse osteoblistic cells was accessed by Alizarin Red staining. The mineralization of MC3T3-E1 cell stimulated with 50 μg/mL T. chebula methanol extract was significantly up-regulated as the day-dependent manner compared with control. Furthermore, the mRNA level of osteocalcin, which is a marker of calcification, was significantly up-regulated by T. chebula methanol extract compared with control. In addition, the mRNA level of collagen Type I and type X, which are a major component of extracellular matrix in the osteoblast, were increased by T. chebula methanol extract as the dose-dependent manner in the MC3T3-E1 mouse osteoblastic cells.

These results showed that the crude extracts of T. chebula Retzius roots could be use as a useful material for the osteoblast cell differentiation and calcification in osteoblast cells. Furthermore, this study suggested that the extracts of T. chebula can use as a natural material of functional foods for the osteoporosis. However, biological compounds with bone formation should be identified through more research in the future.
Alternative Title
Osteogenic effect of Terminalia cebula Retzius extract in pre-osteoblastic cell, MC3T3-E1
Alternative Author(s)
조선대학교 산학협력단
일반대학원 치의생명공학과
Awarded Date
Table Of Contents
제 1장. 서 론 1

제 2장. 연구재료 및 방법 5
1. 실험 재료 및 시약 5
2. 가자 추출물의 제조 5
3. 조골세포 및 정상세포의 배양 6
4. 조골세포의 증식률 및 세포 독성 분석 6
5. Alkaline phosphatase(ALP) 활성 측정 7
6. Collagen 함량 측정 7
7. Mineralization 측정 8
8. Total RNA 분리 및 정량 9
9. cDNA 제조 9
10. PCR(Polymerase Chain Reaction) 분석 9
11. 통계처리 10

제 3장. 연구결과 12
1. 추출물의 세포 독성 분석 12
2. Alkaline phosphatase(ALP) 활성 측정 14
3. Mineralization 측정 16
4. Collagen 함량 측정 18

제 4장. 결론 및 고찰 20
제 5장. 참고문헌 22
박상연. (2013). MC3T3-E1 조골세포에서 가자 추출물에 의한 골형성능 분석.
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