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제대혈 유래 중간엽 줄기세포의 분리 및 특성 규명

Isolation and characterization of mesenchymal stem cells derived from umbilical cord blood

대한산부인과학회지 2006년 49권 5호 p.1073 ~ 1084
김명희/Kim MH
박진현/신찬수/김희중/김정구/Park JH/Shin CS/Kim HJ/Kim JG

Abstract

목적: 제대혈 내 중간엽 줄기세포의 분리법을 개발하고 특성을 규명하고자 하였다.

연구 방법: 제대혈로부터 중간엽 줄기세포를 분리하여 제대혈 중간엽 줄기세포의 증식력, 세포주기, 세포화학 지침자, 면역표현형의 발현도를 측정하였고 골아세포로의 분화도와 지방세포로의 분화도를 조사 분석하였다.

결과: 제대혈로부터 분리된 중간엽 줄기세포의 84% 이상이 세포주기 G0/G1기에 있었고 α-naphthyl acetate esterase, periodic acid-Schiff에 양성반응을 보였으나 sudan black-B, ALP에 음성반응을 보였다. 이들 줄기세포는 CD13, CD29, CD44, CD49e, CD51, CD54, CD90, SH2, SH3 항원을 발현하였으나 CD11b, CD14, CD31, CD34, CD45, CD49d, CD106, CD133, CD144, CD146, CD163, CD166, Stro-1 항원은 발현하지 않았다. 골생성 배양액에서 배양 시 골아세포의 지침자인 ALP, Runx-2, 제 1형 procollagen의 mRNA가 발현되었고 칼슘의 축적 및 ALP활성이 관찰되었다. 지방세포 형성 배양액에서 배양 후 지질축적이 관찰되었다.

결론: 제대혈 내 골형성 및 지방세포로 분화능을 가진 줄기세포가 존재함을 확인하였으며 향후 줄기세포 이용의 잠재적 근원으로 제대혈의 이용 가능성을 시사한다.

Objective: The purposes of this study were to isolate and characterize mesenchymal stem cells (MSCs) with osteogenic and adipogenic potential from umbilical cord blood (UCB).

Methods: MSCs were isolated using a density gradient centrifugation and extensive subcultivation from UCB. The proliferation capability, cell cycle, cytochemical markers, and immunophenotype of these MSCs were measured. The transcript of osteoblast-specific markers, alkaline phosphatase (ALP), and calcium deposit were analyzed from MSCs cultured in osteogenic media for 1-4 weeks, and MSCs exposed in adipogenic media were stained by Oil red after 4 weeks of culture.

Results: More than 84% of MSCs were in the G0/G1 phase of cell cycle. MSCs were positive for periodic acid-Schiff, and -naphthyl α acetate esterase activity, but negative for sudan black-B, and ALP activities. MSCs expressed CD13, CD29, CD 44, CD49e, CD51, CD54, CD 90, SH2, and SH3, but did not express CD11b, CD14, CD31, CD34, CD45, CD49d, CD106, CD133, CD144, CD146, CD163, CD166, and Stro-1. When MSCs were cultured in osteogenic media for 1-4 weeks, they expressed transcripts of osteoblastic specific markers: Runx-2, ALP, and procollagen type I. During culture of 2-4 weeks, ALP activity was detected and quantification increased significantly, and the deposition of a calcified matrix became evident. Exposure of MSCs to adipogenic media resulted in morphological change stained by Oil Red O.

Conclusion: These data indicate that UCB contains MSCs with osteogenic and adipogenic differentiation potential, and may serve as an potential source of MSCs to be utilized in cell therapy for various diseases.

키워드

제대혈;중간엽 줄기세포;특성;Umbilical cord blood;Mesenchymal stem cell;Characteristics
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