혈관내피세포 채취의 원천으로 인간 지방조직의 활용
Use of Human Adipose Tissue as a Source of Endothelial Cells
박봉욱, 하영술, 김진현, 조희영, 정명희, 김덕룡, 김욱규, 김종렬, 장중희, 변준호,
소속 상세정보
박봉욱 ( Park Bong-Wook ) - 경상대학교 의학전문대학원 구강악안면외과학교실
하영술 ( Hah Young-Sool ) - 경상대학교병원 임상의학연구소
김진현 ( Kim Jin-Hyun ) - 경상대학교병원 임상의학연구소
조희영 ( Cho Hee-Young ) - 경상대학교병원 임상의학연구소
정명희 ( Jung Myeong-Hee ) - 경상대학교병원 임상의학연구소
김덕룡 ( Kim Deok-Ryong ) - 경상대학교 의학전문대학원 생화학교실
김욱규 ( Kim Uk-Kyu ) - 부산대학교 치의학전문대학원 구강악안면외과학교실
김종렬 ( Kim Jong-Ryoul ) - 온종합병원
장중희 ( Jang Jung-Hui ) - 경상대학교 의학전문대학원 구강악안면외과학교실
변준호 ( Byun June-Ho ) - 경상대학교 의학전문대학원 구강악안면외과학교실
KMID : 0360120100320040299
Abstract
Purpose: Adipose tissue is located beneath the skin, around internal organs, and in the bone marrow in humans. Its main role is to store energy in the form of fat, although it also cushions and insulates the body. Adipose tissue also has the ability to dynamically expand and shrink throughout the life of an adult. Recently, it has been shown that adipose tissue contains a population of adult multipotent mesenchymal stem cells and endothelial progenitor cells that, in cell culture conditions, have extensive proliferative capacity and are able to differentiate into several lineages, including, osteogenic, chondrogenic, endothelial cells, and myogenic lineages.
Materials and Methods: This study focused on endothelial cell culture from the adipose tissue. Adipose tissues were harvested from buccal fat pad during bilateral sagittal split ramus osteotomy for surgical correction of mandibular prognathism. The tissues were treated with 0.075% type I collagenase. The samples were neutralized with DMEM/and centrifuged for 10 min at 2,400 rpm. The pellet was treated with 3 volume of RBC lysis buffer and filtered through a 100 μm nylon cell strainer. The filtered cells were centrifuged for 10 min at 2,400 rpm. The cells were further cultured in the endothelial cell culture medium (EGM-2, Cambrex, Walkersville, Md., USA) supplemented with 10% fetal bovine serum, human EGF, human VEGF, human insulin-like growth factor-1, human FGF-β, heparin, ascorbic acid and hydrocortisone at a density of 1 × 105 cells/well in a 24-well plate. Low positivity of endothelial cell markers, such as CD31 and CD146, was observed during early passage of cells.
Results: Increase of CD146 positivity was observed in passage 5 to 7 adipose tissue-derived cells. However, CD44, representative mesenchymal stem cell marker, was also strongly expressed. CD146 sorted adipose tissue-derived cells was cultured using immuno-magnetic beads. Magnetic labeling with 100 μl microbeads per 108 cells was performed for 30 minutes at 4℃ a using CD146 direct cell isolation kit. Magnetic separation was carried out and a separator under a biological hood. Aliquous of CD146+ sorted cells were evaluated for purity by flow cytometry. Sorted cells were 96.04% positivity for CD146. And then tube formation was examined. These CD146 sorted adipose tissue-derived cells formed tube-like structures on Matrigel.
Conclusion: These results suggest that adipose tissue-derived cells are endothelial cells. With the fabrication of the vascularized scaffold construct, novel approaches could be developed to enhance the engineered scaffold by the addition of adipose tissue-derived endothelial cells and periosteal-derived osteoblastic cells to promote bone growth.
키워드
Adipose tissue; Endothelial cells; Adipose tissue-derived endothelial cells
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