Chinese Journal of Tissue Engineering Research ›› 2014, Vol. 18 ›› Issue (32): 5203-5208.doi: 10.3969/j.issn.2095-4344.2014.32.020
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Li Zhi1, Zhao Wei1, Liu Wei2, Zhou Ye2, Jia Jing-qiao3, Yang Li-feng1
Received:
2014-06-25
Online:
2014-08-06
Published:
2014-09-18
Contact:
Yang Li-feng, Department of Orthopedics, Central Hospital of Shenyang Medical University, Shenyang 110024, Liaoning Province, China
About author:
Li Zhi, Studying for doctorate, Chief physician, Department of Orthopedics, Central Hospital of Shenyang Medical University, Shenyang 110024, Liaoning Province, China
Supported by:
the Scientific Research Program of Liaoning Science and Technology Bureau, No. 2012225014
CLC Number:
Li Zhi, Zhao Wei, Liu Wei, Zhou Ye, Jia Jing-qiao, Yang Li-feng . Chondrogenic differentiation of placenta-derived mesenchymal stem cells in vitro[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(32): 5203-5208.
2.1 胎盘来源间充质干细胞形态及细胞表面标志物检测结果 人胎盘间充质干细胞在间充质干细胞培养基中培养,细胞增殖形成大量贴壁细胞,放大40倍倒置显微镜观察,贴壁细胞具有典型的间充质细胞形态,即纺锤体型,折光性强。 用流式细胞仪检测结果显示,阳性表达CD90、CD44、CD105和CD73,不表达CD34、CD14和CD45。流式细胞仪检测结果表明实验培养的细胞具备干细胞的表面标志物,可证实为胎盘间充质干细胞。 2.2 成软骨细胞诱导后阿利新蓝染色结果 倒置显微镜观察:对照组1:形成微团时所用的胎盘来源间充质干细胞的细胞悬液体积为5 μL,细胞悬液浓度为1.6×1010 L-1,在间充质干细胞培养基中培养,每3 d换液1次。细胞增殖形成大量贴壁细胞,14 d后阿利新蓝染色,放大40倍倒置显微镜观察,贴壁细胞具有典型的间充质细胞形态,即纺锤体型,折光性强,色成淡蓝色(图1A)。 对照组2:形成微团时用细胞浓度为1.6×1010 L-1的胎盘来源间充质干细胞悬液10 μL,可见细胞增殖形成大量贴壁细胞外,还会形成细胞膜状反折,14 d后阿利新蓝染色,倒置显微镜观察,贴壁细胞具有典型的间充质细胞形态(图1B)。 对照组3:形成微团时用细胞浓度为1.6×1010 L-1的胎盘来源间充质干细胞悬液15 μL,4 d后行阿利新蓝染色,倒置显微镜观察,微团的基部具有大量贴壁细胞,微团不圆润(图1C)。 实验组1:形成微团时所用的胎盘来源间充质干细胞的细胞悬液体积为5 μL,细胞悬液浓度为1.6×1010 L-1,在软骨诱导培养基中培养,每3 d换液1次。14 d后行阿利新蓝染色,放大40倍倒置显微镜观察,细胞只会保持微团,不会继续增殖形成贴壁细胞,微团基部无贴壁细胞,微团悬浮培养基中,微团直径约1 mm。倒置显微镜下无法观察到微团内的细胞形态,但可观察到实验组中微团的边缘向外延伸出少数的丝状细胞结构(图1D)。 实验组2,3:形成微团时用细胞浓度为1.6×1010 L-1的胎盘来源间充质干细胞悬液10,15 μL,倒置显微镜观察悬浮培养基中的微团直径约1.5,2 mm,其形态与实验组1相似(图1E,F)。 大体观察结果:各组胎盘来源间充质干细胞的数码相机照相结果见图2。 对照组1,2中细胞在间充质干细胞培养基中增殖良好,形成大量贴壁细胞长满24孔板(图2A,B);对照组3中有细胞团出现(图2C);实验组细胞在软骨诱导培养基中培养,未见贴壁细胞,微团直径约1.0,1.5,2.0 mm(图2D-F)。 "
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