CAJ | 학술논문

目的研究人体瘢痕疙瘩来源的前体细胞(keloid-derived precursor cell,KPC)的体外分离、扩增方法,及其在特定培养基条件下向脂肪细胞的诱导分化,探讨其作为组织工程脂肪种子细胞的可能性.方法取人体瘢痕疙瘩组织在改良L-DMEM培养基条件下培养,传代后以流式细胞仪检测其间充质干细胞(mesenchymal stem cell,MSC)相关基因表达,并以特定H-DMEM培养基[含1 μmol/L的地塞米松,0.5 mmol/L的3异丁基-1甲基黄嘌呤(3-IBMX),10 mg/L牛胰岛素,100 mmol/L的消炎痛,10％的胎牛血清(FBS)]诱导,在相差显微镜下观察,油红O染色测定脂滴生成.结果超过95％的KPC表达CD29、CD44、CD90/Thy-1和CD105;几乎不表达CD45和CD34(1.0％～2.5％)；且诱导后该细胞渐增大,由梭形变为圆形或多角形,72 h出现脂滴,19d油红O染色细胞阳性率78.6％.结论 KPC能表达多种MSC表面标志物,不表达造血干细胞(hemopoietic stem cell,HSC)标志物,能够在诱导培养基条件下分化为脂肪细胞,可能成为组织工程脂肪的一种新种子细胞来源.
목적 연구인체반흔흘탑래원적전체세포(keloid-derived precursor cell,KPC)적체외분리、확증방법,급기재특정배양기조건하향지방세포적유도분화,탐토기작위조직공정지방충자세포적가능성.방법 취인체반흔흘탑조직재개량L-DMEM배양기조건하배양,전대후이류식세포의검측기간충질간세포(mesenchymal stem cell,MSC)상관기인표체,병이특정H-DMEM배양기[함1 μmol/L적지새미송,0.5 mmol/L적3이정기-1갑기황표령(3-IBMX),10 mg/L우이도소,100 mmol/L적소염통,10％적태우혈청(FBS)]유도,재상차현미경하관찰,유홍O염색측정지적생성.결과 초과95％적KPC표체CD29、CD44、CD90/Thy-1화CD105;궤호불표체CD45화CD34(1.0％～2.5％)；차유도후해세포점증대,유사형변위원형혹다각형,72 h출현지적,19d유홍O염색세포양성솔78.6％.결론 KPC능표체다충MSC표면표지물,불표체조혈간세포(hemopoietic stem cell,HSC)표지물,능구재유도배양기조건하분화위지방세포,가능성위조직공정지방적일충신충자세포래원.
Objective To explore the isolation,amplification methods and adipogenic differentiation under specific culture medium of human keloid-derived precursor cell (KPC) in vitro,in order to study their possibility of being new seed cells of tissue engineering fat.Methods KPCs were isolated from human keloid tissue of 4 different patients in our hospital and were cultured in the modified L-DMEM culture medium.Their cloning efficiency and growth curve were tested.The subcultured cells were tested of the mesenchymal stem cell (MSC)-related gene expression by flow cytometry.In addition,they were cultured in H-DMEM medium (containing 1 μmol/L dexamethasone,0.5 mmol/L 3-isobutyl-1-methyl-xanthine 10 mg/L of bovine insulin,100 mmol/L indomethacin,and 10 ％ FBS)and were later observed in oil red O staining under phase contrast microscope to determine whether lipid droplets generation was formed,using skin-derived precursors (SKP) as control.Results More than 95 ％ KPC expressed many antigens of MSC,such as CD29,CD44,CD90 and CD105 while few of them expressed CD34,CD45(1.0 ％-2.5 ％).And the cells increased in size gradually after inducted the same time,changing from spindle into round or polygonal in shape.The lipid droplets were seen in 72 hours and expressed a positive rate of 78.6 ％ in Day 19 in oil red O staining while the same rate was 54.6 ％ in SKP.Conclusions Human keloid-derived precursor cells can express a variety of MSC-related surface markers without expressing hematopoietic stem cell (HSC) related markers.Furthermore,they can be differentiated into fat cells under certain conditions,which may make them as a new source of seed cells for tissue engineering fat.