초록 ▼

1. 인간 치주인대 세포주 Bank 설립
- 동의서 확보, 치주인대세포 배양, 저장용기 확보 및 database화

2. 치주인대세포 채득방법 확립 및 치주인대세포의 광물화 형성능 규명
- explant & enzyme 기술을 이용하여 치주인대세포 획득 및 광물화 형성 평가

3. 치주인대세포의 백악아세포 및 섬유아세포로의 분화 관련 유전자 규명
- cDNA microarray를 이용하여 분화 관련 유전자 발굴
- BMP2와 FGF2에 대한 관련 유전자의 반응 평가

4. 치주인

1. 인간 치주인대 세포주 Bank 설립
- 동의서 확보, 치주인대세포 배양, 저장용기 확보 및 database화

2. 치주인대세포 채득방법 확립 및 치주인대세포의 광물화 형성능 규명
- explant & enzyme 기술을 이용하여 치주인대세포 획득 및 광물화 형성 평가

3. 치주인대세포의 백악아세포 및 섬유아세포로의 분화 관련 유전자 규명
- cDNA microarray를 이용하여 분화 관련 유전자 발굴
- BMP2와 FGF2에 대한 관련 유전자의 반응 평가

4. 치주인대세포의 백악아세포로의 분화 관련 유전자 기능 분석을 위한 virus 구축

5. 치주인대세포의 골 조직 재생능을 누드마우스의 두개골 결손부 모델을 통해 확인

6. C57BL/6 생쥐 유래 배아 줄기 세포 유도

7. 줄기세포 배양을 위한 새로운 feeder 세포 탐색

8. 치아 수 결정 유전자 Runx2 와 Ectodin의 관계규명

9. 사람의 치아결손 질환자에서 유전자 발현조사

10. static tensional force에 의한 치주 인대 세포의 유전자 발현 변화 규명

(출처 : 보고서 요약서 3p)

Abstract ▼

We were received a patient written consent from patients. After, the PDL tissues were obtained from the periodontal ligament of premolar teeth extracted for orthodontic reasons. Normal impacted premolars were collected from 29 donors at the Department of Periodontology, National University Hospital.

We were received a patient written consent from patients. After, the PDL tissues were obtained from the periodontal ligament of premolar teeth extracted for orthodontic reasons. Normal impacted premolars were collected from 29 donors at the Department of Periodontology, National University Hospital. We secured 62 hPDL cells bank. And also, we checked the degree of mineralization and the osteoblastic-differentiation related markers in the hPDL cells isolated by explant or enzyme technic. We confirmed that hPDL cells isolated by explant technic were more high ALP activiy, mineralized nodule formation than the hPDL cells isolated by enzyme technic. But, proliferation is higher in hPDL cells isolated by enzyme technic than hPDL cells isolated by explant technic. Also, we confirmed that hPDL cells expressed stem cell marker such as CD146, STRO-1.

Next, we observed the gene expression profile in hPDL cells proliferation, differentiation and mineralization in vitro. At day 1 and 7, any mineralization nodule was not observed. A few, small mineralized nodules started to be developed on day 14, and mineralization was distinct. on day 21.

A number of mineralization nodules were formed and mineralization nodule size was also increased on day 21. At day 0 showing confluent monolayer, as a active proliferative stage, C-myc gene expression observed maximal level.

At day 7 showing multilayer formation, alkaline phosphatase, BMP-2 and BMP-4 gene expression was increased and followed by maximal expression of osteocalcin on day 14 showing initiation of nodule mineralization. In relation to apoptosis, C-fos gene expression was peaked on day 21 characterized postmineralization stage.

We investigated the up/down-regulated genes in the hPDL cells during mineralization at 7 and 14 days by using DNA microarray. We filtered the genes over the 3-fold change and the signal value 100. At day 7, 111 genes (128 probes) were expressed more than 3 folds in osteogenic medium-hPDL cells, whereas 19 genes (22 probes) were down-regulated less than -3 folds in non-osteogenic medium-hPDL cells. At 14 days, 77 genes (99 probes) were expressed more than 3 folds in osteogenic medium-hPDL cells, whereas 19genes (21 probes) were down-regulated less than -3 folds in non-osteogenic medium-hPDL cell. At both days, 30 genes (45 probes) were up-regulated, whereas 11 genes (14 probes) were down-regulated. We selected top 10 genes that either increased or decreased. And then, we evaluated the response to the highly expressed genes by rhBMP-2 and FGF-2 during mineralization of the hPDL cells. According to concentration of rhBMP-2, PLZF, FKBP5 and FAM107A gene expression showed the increasing tendency, but SAA1, FABP4 and PIP gene expression showed the decreasing tendency. According to exposed time of rhBMP-2, every genes showed the gradually increasing tendency. According to concentration and exposed time of FGF-2, All genes except for RAC3 showed the decreasing tendency.

Thus, these findings demonstrated that the highly expressed genes during mineralization of the hPDL cells were classified by runx-2 activator, NF-kB signaling-related factors and apoptosis-related factors, and responsed reversely to rhBMP-2 and FGF-2. On the base of these results, PLZF, FKBP5, SAA1, FABP4, FAM107A, CORIN, RAC3 and PIP are considered as the osteoblastic/cementoblastic differentiation-related genes in the hPDL cells.
When we transfected PLZF in hPDL cells, bone-related genes such as Runx-2, ALP, BSP expressions were more high than normal hPDL cells.

Thus, PLZF is suggested the cementoblastic differentiation related genes. It has to be studied further to evaluate the role of these genes in the osteoblastic/cementoblastic differentiation in the hPDL cells.
Next, we observed the osteoblastic-regeneration of hPDL cells in vivo.
The Collatape®(Zimmer dental, co, USA) was used for carrier of hPDL cells.

The PDL cells were cultured on the Collatape® in osteogenic medium. At 7 days, the collatape with or without cultured hPDL cells was observed through scanning electron microscope(SEM). And then, we observed the expression of osteoblast-related marker such as Runx-2, ALP, BSP, OCN, CP-23, F-spondin, Col I and Scleraxis between day 0 and 7. At 7 days, the expression of osteoblast-related markers observed more high than Day 0. And then, we translocated the cultured hPDL cells with collatape for 7 days in nude mouse calvarial defects. Only soaked Collatape® in medium for 7 days was used for controls. The defects were evaluated in histologic after 8 weeks. At 8 weeks of healing, new bone formation was found in the defect in test group. However in control group, the defect was packed with fibrous connective tissue. There was not inflammatory reaction in the all groups. In conclusion, we can suggested that hPDL cells can increase new bone formation in nude mouse calvarial defects. On the basis of this results, this new strategy may be applied in the treatment of bony defects of periodontal therapy.

Although most mouse embryonic stem (ES) cells were derived from blastocysts of 129/sv strain, it has been known that this strain has many problems including spontaneous teratoma formation and behavioral and immunological responses. Therefore, it is necessary to backcross gene-manipulated mice derived from this ES cell with inbred C57BL/6 strain to overcome these problems. In this study, we successfully derived mouse ES cells from delayed blastocysts after ovariectomy and injection of progesterone. Total 15 different mouse ES cells were derived from 70 female C57BL/6 mice. Characterization of CJ42-1 ES cells, one of derived ES cells, was performed by analysis of the expression of ES cells markers. We also identified various tissues derived from all three germline layers in excised teratoma by injection of ES cells into nude mice. Collectively, derivation of ES cells from C57BL/6 mice was successfully performed by production of delayed blastocysts.

Mouse ES cells need to culture on feeder cells such as mouse embryonic fibroblasts (MEF). MEF secrete many factors including LIF, BMPs and these factors support to maintain self-renewal and pluripotency of ES cells. MEF has disadvantages like limited passage number. In this study, a new feeder X-1 cells supported to maintain self-renewal and pluripotency of ES cells and overcome a disadvantage of limited passage number of MEF.

One of characteristic features of RUNX2 heterozygous patients is supernumerary teeth. But we did not find this phenotype in mouse Runx2 heterozygote. Ectodin-deficient mice have extra teeth. We try to elucidate relation between Runx2 and Ectodin. In situ hybridazation of Ectodin was decreased in Runx2 heterozygote. We also found that Runx2 didn't bind to ectodin promoter, which was confirmed by Ectodin-Luciferase reporter assay.

These results indicate that Runx2 does not regulate ectodin expression directly in tooth patterning. However, it may be possible that Runx2 can regulate ectodin expression indirectly in tooth patterning.

Furthermore, gene expression patterns in abnormal tooth eruption and in PDL cells applied with static tensional forces were established. Thes results also can help improve the bio-tooth regeneration research.

(출처 : SUMMARY 11p)

목차 Contents

• 표지 ... 1제 출 문 ... 2보고서 요약서 ... 3요 약 문 ... 4SUMMARY ... 11CONTENTS ... 14목차 ... 15제 1 장 연구개발과제의 개요 ... 16제 2 장 국내외 기술개발 현황 ... 18제 3 장 연구개발수행 내용 및 결과 ... 19 3. 1. 치주인대세포의 채득방법에 따른 성상비교 ... 19 3. 2. 치주인대세포의 광물화 형성과정 시 발현되는 다양한 유전자 양상의 검토 ... 21 3.3 치주인대세포의 광물화과정시 발현되는 유전자들에 대한 상호비교 ... 26 3. 4. 치주인대세포의 골아세포 및 백악아세포로의 분화시 강하게 발현되는 PLZF의 효과 ... 32 3.5. rhBMP-2가 고농도 당에서 배양된 치주인대세포의 광물화 형성과정에 미치는 영향 ... 36 3.6. 누드마우스의 두개골 결손부에 배양된 치주인대 세포 이식이 신생골 형성에 미치는 영향 ... 39 3.7. 다양한 세포부착 펩타이드로 흡착된 골이식재가 골아세포 활성도와 골형성에 미치는 영향 ... 41 3.8. C57BL/6 생쥐 유래 배아 줄기 세포 유도 ... 45 3.9. 줄기세포 배양을 위한 새로운 feeder 세포 탐색 ... 48 3.10. 치아수 결정 pathway 규명 ... 49 3.11. 사람의 치아결손 질환자에서 유전자 발현연구 ... 52 3.12. static tensional force에 의한 치주 인대 세포의 유전자 발현 변화 규명 ... 53 3. 13. Anti-allergic effect of Vitis amurensis on mast cell-mediated allergy model. Exp Biol Med. 233(2):192-199. ... 55 3.14. Signaling pathways in bisphenol A-induced apoptosis: role of calcium-induced reactive oxygen species, mitogen-activated protein kinases and nuclear factor-κB. ... 60 3.15. Mosla dianthera decreases immediate-type allergic reaction and tumor necrosis factor-α production. ... 64 3.16. Motherwort의 항염증 및 항알레르기 효과 발굴 ... 67제 4 장 목표달성도 및 관련분야에의 기여도 ... 71제 5 장 연구개발결과의 활용계획 ... 76제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 77제 7 장 참고문헌 ... 78끝페이지 ... 79