초록 ▼

최종 연구결과 요약
◦세부과제명: 개 유래 성체줄기세포 작성, 유지, 성상 및 분화에 관한 연구
1) 개의 제대조직에서 유래된 다능성 성체줄기세포 4주와 지방조직에서 유래된 다능성 성체줄기세포 14주 확립
2) 확립된 성체줄기세포 18주의 세포표면 항원 단백질을 이용한 성상확인
- 양성확인 : CD 44, 54, 61, 80, 90, 105, 184, FLK-1, HMGA2
- 음성확인 : CD 34, 45, 117 또는 MHC-II
3) 확립된 성체줄기세포주의 줄기세포성 확인
- 양성확인 :

최종 연구결과 요약
◦세부과제명: 개 유래 성체줄기세포 작성, 유지, 성상 및 분화에 관한 연구
1) 개의 제대조직에서 유래된 다능성 성체줄기세포 4주와 지방조직에서 유래된 다능성 성체줄기세포 14주 확립
2) 확립된 성체줄기세포 18주의 세포표면 항원 단백질을 이용한 성상확인
- 양성확인 : CD 44, 54, 61, 80, 90, 105, 184, FLK-1, HMGA2
- 음성확인 : CD 34, 45, 117 또는 MHC-II
3) 확립된 성체줄기세포주의 줄기세포성 확인
- 양성확인 : OCT3/4, SOX2, NANOG
4) 확립된 성체줄기세포의 분화유도 확인 : Adipocytes, Osteocytes, chondrocytes 분화됨
5) 개 성체줄기세포의 증식능, 줄기세포성, 세포표면 항원 단백질, 분화능을 계대별 비교
- 계대수가 증가할수록 증식능, 세포표면 항원 단백질, 분화능 감소하거나 사라짐
- 줄기세포성은 계대수가 증가하여도 약간 감소함◦ 세부과제명 : 개의 질병 치료 적용을 위한 줄기세포 성상 비교
- 7개월령 개체와 10-11살 개체 유래의 성체줄기세포의 증식능, 줄기세포성, 세포표면 항원 단백질, 분화능을 계대별 비교한 결과, 10-11살 개 유래의 줄기세포가 7개월령 개 유래의 줄기세포 보다 줄기세포성이나 세포표면 항원 단백질의 발현량 및 증식능과 분화능이 낮음을 확인할 수 있었음. 이를 바탕으로 어린 개체의 줄기세포를 임상에 적용하는 것이 바람직하다고 판단함
◦ 세부과제명 : 줄기세포를 이용한 개의 질병 치료 적용
1) 단백소실성 신장질환 및 만성 단백소실성 장염의 환축에 2주 간격 19회 줄기세포 적용
- 피하주사로 줄기세포를 투여한 결과 설사증상이 완화
- 그 이후에는 4주 간격 6회 추가 적용
- 줄기세포를 지속적으로 적용한 결과, 저알부민혈증의 개선 관찰 : 혈중 알부민 수치 상승(1.9→2.4), 체중 증가 (1.2→1.44kg) 확인
2) 관절염 환축에 2달 간격으로 21개월 가량 (8회) 줄기세포를 지속적으로 투여
- 골밀도 증가 관찰, 통증 감소효과를 확인하였고, 보행상태가 호전됨

Abstract ▼

Effects of serial passage on the characteristics and chondrogenic differentiation of canine umbilical cord matrix derived esenchymal stem cells:
Mesenchymal stem cells (MSCs) are often known to have therapeutic potential in the cell-mediated repair for fatal or incurable diseases.In this study,

Effects of serial passage on the characteristics and chondrogenic differentiation of canine umbilical cord matrix derived esenchymal stem cells:
Mesenchymal stem cells (MSCs) are often known to have therapeutic potential in the cell-mediated repair for fatal or incurable diseases.In this study, canine umbilical cord MSCs (cUC-MSCs) were isolated from umbilical cord matrix (n=3) and subjected to proliferative culture for 5 consecutive passages. The cells at each passage were characterized for multipotent MSC properties such as proliferation kinetics, expression patterns of MSC surface markers and self-renewal associated markers, and chondrogenic differentiation. In results, the proliferation of the cells as determined by cumulative population doubling level were observed at its peak on passage 3 and stopped after passage 5, whereas cell doubling time dramatically increased after passage 4. Expression of MSC surface markers (CD44, CD54, CD61, CD80, CD90 and Flk-1), molecule
(HMGA2) and pluripotent markers (sox2, nanog) associated with self-renewal was negatively correlated with the number of passages. However, MSC surface markers (CD105), pluripotent marker (Oct3/4) was decreased with increasing the number of
subpassage. cUC-MSCs at passage1 to 5 underwent chondrogenesis under specific culture conditions, but percentage of chondrogenic differentiation decreased with increasing the number of subpassage. Collectively, the present study suggested that sequential subpassage could affect multipotent properties of cUC-MSCs and it needs to be addressed before clinical applications.

Sequential sub-passage lowers the differentiation potential of canine adipose derived mesenchymal stem cells:
Adipose-derived mesenchymal stem cells (AD-MSCs) have demonstrated the feasibility of the usage in clinical application, due to ease of isolation and abundance in the tissue without age barrier with the great potential of differentiation into multi-lineage cells. However, the promising potential only be achieved if the cells are expanding in a large number while maintaining their stemness in sequential passages. Inthis study, canine AD-MSCs were isolated (n=5) and subjected to proliferative culture with 7 sub-passages. The cells at each subpassge were characterized for properties associated with multipotent MSCs such as proliferation kinetics, expression of MSCs-specific surface markers, expression of molecules associated with self-renewal and differentiation capabilities into mesodermal lineage cells. In results, the proliferation of the cells was plateaued at passage 5 by cumulative population doubling level, while cell doubling time gradually increased with passage. MSCs surface markers (CD44, CD90, and CD105) and molecules (Oct 3/4, Sox-2, Nanog and HMGA2) associated with self-renewal were all expressed in the cells between passages 1 to 6 by RT-PCR. In addition, the cells at passage 1, 3 or 6 underwent adipogenic and chondrogenic differentiation under specific induction conditions. However, the level of adipogenic and chondrogenic differentiation was negatively correlated with the number of sub-passage. In conclusion, the present study suggests that sequential sub-passages affect multipotent properties of cAD-MSCs, which should be considered their therapeutic application in regenerative medicine.

목차 Contents

• 연구과제 최종결과보고서 ... 1
• Ⅰ. 연구배경 및 목표 ... 3
• 1. 연구배경 ... 3
• 2. 연구최종목표 ... 4
• 3. 연차별 연구개발 목표 및 내용 ... 4
• Ⅱ. 연구방법 및 수행전략 ... 5
• 1. 재 료 ... 5
• 2. 연구전략 및 방법 ... 5
• Ⅲ. 연구결과 ... 7
• □ 1년차 (2011) ... 7
• □ 2년차 (2012) ... 10
• □ 3년차 (2013) ... 14
• Ⅳ. 연구결과요약 ... 23
• Ⅴ. 고 찰 ... 24
• Ⅵ. 연구결과 활용실적 및 계획 ... 24
• Ⅶ. 연구사업 추진상 문제점 및 건의사항 ... 25
• Ⅷ. 주요연구과제 변경사항 ... 25
• Ⅸ. 참고문헌 ... 25
• Ⅹ. 영문초록 ... 26
• 끝페이지 ... 27