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말 관절염 치료를 위한 말 성체줄기세포 배양 및 규명에 관한 연구

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Author(s)
진남일
Issued Date
2010
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000005115
Abstract
배경: 말은 이미 연골손상과 골관절염에 초점을 둔 동물모델로 자리 잡고 있다. 다른 동물과 비교했을 때 말 관절모델의 장점은 조작과 탐구에 유리한 관절의 크기와 현재의 동물모델들 사이에서 인간의 관절연골과 가장 닮은 연골두께와 구성에 있다. 사람과 말의 아킬레스건(Achilles tendon)과 같은 체중부하힘줄(weight-bearing tendon) 사이에는 계층구조, 기질구성, 기능 그리고 지속되는 손상의 본질에 있어서 비슷한 점이 많다. 본 연구에서는 이와 같이 관절염 치료 모델로서의 많은 장점을 가지고 있는 말의 골수유래 줄기세포를 분리하여 연골세포 특이 유전자의 발현양을 확인하고 관절염을 유발시킨 마우스의 무릎 관절강 내로 말 골수 줄기세포를 주사하여 줄기세포의 연골재생 치료 효과를 알아보고자 실험을 실시하였다.

재료 및 방법: 본 연구에서는 제주축산진흥원으로부터 말 골수를 분양받아 실험에 사용하였다. 분양받은 골수는 원심분리하여 단핵세포를 분리 배양하였고, 첫 번째 계대배양 후 얻어진 골수유래 줄기세포는 haemacytometer를 사용하여 counting한 다음 MTT 검사법을 이용하여 세포 증식률을 측정하였다. 배양중인 줄기세포의 분화능을 확인하기 위해 Oil red-O staining과 H&E staining을 실시한 후 광학현미경 하에서 관찰하였으며, RT-PCR을 실시하여 분화유도 배지에서 배양한 줄기세포의 collagenⅠ, collagenⅡ, PPARγ1, Oct4 유전자에 대한 발현 양상을 관찰하고자 하였다. 또한 관절염이 유발된 마우스의 무릎 관절강 내로 말 골수유래 줄기세포를 투여한 후 인대와 건 주위의 세포를 추출하여 TGF-β1, TGF-β2, TGF-β3, MMP2 유전자의 시간에 따른 발현양을 RT-PCR로 관찰하였다. GFP 유전자로 전이된 말 골수유래 줄기세포를 면역억제제를 주사한 마우스의 생체 내로 투여하여 생체내 여러 기관으로 전이되는지 형광현미경 하에서 관찰하였으며, 관절염이 유발된 마우스에 말 골수유래 줄기세포를 투여 했을 때 마우스의 관절염 회복에 미치는 영향에 관해서 알아보고자 하였다. 관절염 유발 마우스의 회복률은 면역억제제를 처리한 마우스 군과 면역억제제를 처리하지 않은 마우스 군으로 나누어 말 골수유래 줄기세포를 투여한 후 관절염을 유발시켰을 때 마우스의 움직임과 비교하여 분석 하였다.

결과: 본 연구에서 분리된 단핵세포는 배양접시 바닥에 부착되어 집락을 형성하며 성장하였으며, 배양 후 3일부터 방추형의 긴 모양인 섬유아세포의 형태를 보였다. 말 골수유래 줄기세포의 증식속도는 day 4일부터 day 5일 사이에 약 5배로 증식하여 배양 기간 중 가장 높은 증식률을 보였으며, 그 이후에는 세포증식률이 낮아져 비교적 일정한 속도로 증식하는 것을 알 수 있었다. RT-PCR을 통한 유전자의 발현양 검사에서 collagenⅠ의 발현은 분화 초기에 높게 나타났으며, collagenⅡ의 발현은 초기 이후에 높게 나타났다. PPARγ1의 발현은 시간의 경과에 따른 유의적인 차이 없이 아주 미약하였고, Oct4의 발현은 일반 배지에서 배양한 줄기세포에서는 발현되었으나, 분화유도 배지에서 배양한 줄기세포에서는 발현되지 않아 분화가 잘 되었음을 알 수 있었다. 관절염이 유발된 마우스의 무릎 관절강 내로 말 골수유래 줄기세포를 투여한 후 마우스의 인대와 건주위의 세포를 추출하여 RT-PCR을 통하여 TGF-β1, TGF-β2, TGF-β3, MMP2 유전자의 시간에 따른 발현양을 관찰하였다. 그 결과 연골세포의 분화에 중요한 유전자인 TGF-β(transforming growth factor beta, TGF-β1, 2, 3)는 시간의 경과에 따라 발현양이 다르게 나타났는데, TGF-β1은 첫째 달에 높은 발현율을 보여 연골분화 초기에 중요한 작용을 하는 것으로 나타났고, TGF-β2와 TGF-β3는 셋째 달에 높은 발현율을 보여 연골분화 초기 이후에 중요한 작용하는 것으로 나타났다. 연골기질 분해 효소인 MMP2의 경우는 시간의 경과에 따른 유의적 차이는 없었으며 발현의 정도는 아주 미약한 것으로 나타났다. 관절염이 유발된 마우스의 무릎 관절강 내로 말 골수유래 줄기세포를 이식하여 육안으로 관찰한 결과 면역억제를 처리한 마우스에 줄기세포를 투여한 군과 면역억제제를 처리하지 않은 마우스에 줄기세포를 투여한 군 모두에서 관절염을 유발시켰을 때보다 뒷다리의 움직임이 현저히 완화되어 높은 회복률을 보였다. 하지만 면역억제제를 처리한 군과 면역억제제를 처리하지 않은 군 사이에서의 움직임에는 별다른 차이가 없었다.

결론: 본 연구에서는 마우스에 관절염을 유발시켜 말 골수유래 줄기세포를 주입한 결과 줄기세포를 주입한 실험군은 줄기세포를 주입하지 않은 대조군에 비하여 높은 회복률을 보였다. 하지만 면역억제제를 처리한 마우스에 줄기세포를 주입한 군과 면역억제제를 처리하지 않은 마우스에 줄기세포를 주입한 군 사이에서는 별다른 차이가 없었다. 이러한 결과로 볼 때 줄기세포 치료를 위해 이종의 줄기세포를 이용하여도 치료의 효과가 나타날 수 있음이 사료된다. 하지만 육안관찰을 통하여 회복률을 평가하였기에 정확성에 한계가 있으므로 차후 좀 더 과학적인 장비를 이용하여 분석하는 과정이 필요하다고 하겠다.
Purpose: The horse is already established as an animal model for focal cartilage injuries and osteoarthritis. Advantage of horse joint model compared with those of other animals are their sheer size, which allows for easy manipulation and exploration, and their cartilage thickness and composition, which most closely resemble those of human articular cartilage among the current animal models. There are many similarities between the weight-bearing tendons of the horse and those of the human achilles tendon in their hierarchical structure and matrix composition, their function and the nature of the injuries sustained. This research was based on isolation stem cells from bone marrow of horse which had many advantage as an arthritis treatment model, and checking a expression of the specific gene of cartilage cells and testing a effectiveness of cartilage repair treatment using stem cells by injecting with horse bone marrow stem cells(BMSCs) into the mouse which was induced arthritis.

Materials and Methods: The horse bone marrow was brought from Jeju livestock institute for this research. Mono nuclear cells were isolated from the horse bone marrow using centrifuge and cultured cells. Bone marrow stem cells(BMSCs) which is gotten after first subculture was counted using haemacytometer and then measured rate of cell multiplication using MTT assay. The cells that Oil red-O and H&E staining was done for checking a differentiation potential culturing stem cells were observed through an optical microscope and RT-PCR was carried out for observing a distribution volume of collagenⅠ, collagenⅡ, PPARγ1 and Oct4 gene using stem cells which was cultured in medium with differentiation inducers. In addition RT-PCR was carried out for observing a expression as time duration of TGFβ-1, TGFβ-2, TGFβ-3 and MMP2 gene using a nearby cells of a part of ligament and tendon of the mouse which was induced arthritis and injected the horse bone marrow derived stem cells. The horse bone marrow derived stem cells which is transfected with GFP vector are injected into mouse which was injected with immunosuppressive agents and then observed through a fluorescence microscope for checking the injected stem cells whether or not move to various organs, and it was checked to influence recovery of arthritis when the horse bone marrow derived stem cells were injected into the mouse which was induced arthritis. The recovery rate of the mouse which was induced arthritis was compared injecting a horse bone marrow derived stem cells with immunosuppressive agents group and a horse bone marrow stem cells with no immunosuppressive agents group with the control group when the mouse was induced arthritis.

Results: The isolated mono nuclear cells in this research grew with colony formation on surface of a bottom of flask, and after three days the cells were come into sight fibroblast-like shape of shpindle-shaped. Doubling time of the horse bone marrow derived stem cells was highest from day 4 to day 5 as five times, and after that a multiplication rate was went down and the multiplication rate kept relatively consistent speed. In a gene expression test through RT-PCR, the expression of collagenⅠwas early differentiation and the expression of collagenⅡ was high after early differentiation and the expression of PPARγ1 was very low with no difference as time duration and Oct4 was expressed on stem cells which is cultured in normal medium and Oct4 was not expressed on stem cells which is cultured in medium with differentiation inducers though. Therefore stem cells which is cultured in medium with differentiation inducers were well differentiated. The result of observing a expression as time duration of TGFβ-1, TGFβ-2, TGFβ-3 and MMP2 gene using a nearby cells of a part of ligament and tendon of the mouse which was induced arthritis and injected the horse bone marrow derived stem cells, a expression of TGF-β(transforming growth factor beta, TGF-β1, 2, 3) which is important gene for differentiation of cartilage cells was different as time duration. TGFβ-1 was important effect early cartilage differentiation because the expression of TGFβ-1 was high after one month, and TGFβ-2 and TGFβ-3 were important effect after early cartilage differentiation because the expression of TGFβ-2 and TGFβ-3 were high after three months. The expression of MMP2 was very low with no difference as time duration. The result of observing with the unaided eye after injecting the horse bone marrow derived stem cells into the mouse which was induced arthritis, both injecting a horse bone marrow derived stem cells with immunosuppressive agents group and a horse bone marrow derived stem cells with no immunosuppressive agents group were noticeable in a recovery rate compared the control group when the mouse was induced arthritis. But it was no noticeable difference between injecting a horse bone marrow derived stem cells with immunosuppressive agents group and a horse bone marrow derived stem cells with no immunosuppressive agents group in a recovery rate.

Conclusion: In this research, the result of observing after injecting the horse bone marrow derived stem cells into the mouse which was induced arthritis, the injecting a horse bone marrow derived stem cell group was noticeable in a recovery rate compared the control group which is not injected a horse bone marrow derived stem cells. But it was no noticeable difference between injecting a horse bone marrow derived stem cells with immunosuppressive agents group and a horse bone marrow derived stem cells with no immunosuppressive agents group in a recovery rate. As the result of an experiment, it is possible to use heterogeneity stem cells for stem cell therapy. But it was limited as accuracy because of observing with the unaided eye, therefore it is need to analyze with scientific exactitude in the future.
Alternative Title
The study on the investigation and equine adult stem cell culture in therapy for equine arthritis
Affiliation
제주대학교 산업대학원
Department
산업대학원 농업생명과학과
Advisor
정동기
Awarded Date
2010. 8
Table Of Contents
목 차


ABSTRACT ································································· 1

Ⅰ. 서론 ···································································· 4

Ⅱ. 연구사 ································································· 7

Ⅲ. 재료 및 방법 ················································· 13

1. 실험동물 ·······················································13
2. 줄기세포 분리 및 배양 ········································13
3. 세포증식률 측정 ···············································14
4. 분화능 관찰 ···················································14
5. RT-PCR ·······················································15
6. GFP 유전자 전이 및 생체내 이식 ····························18
7. 관절염 유발 및 줄기세포 이식 ·······························18

Ⅳ. 결과 ···························································· 20

Ⅴ. 고찰 ···························································· 29

Ⅵ. 요약 ···························································· 32

참고문헌 ··························································· 34
Degree
Master
Publisher
제주대학교 산업대학원
Citation
진남일. (2010). 말 관절염 치료를 위한 말 성체줄기세포 배양 및 규명에 관한 연구
Type
Dissertation
Appears in Collections:
Graduate School of Industry > Ecofriendly Agricultural Science
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