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人羊膜来源间质干细胞的分离、培养及鉴定

Isolation,culture and identification of human amniotic-derived mesenchymal stem cells

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【作者】 李国喜； 杨波； 关方霞； 杜英； 宋来君； 张清勇； 胡祥； 胡平生； 徐虹；

【Author】 LI Guoxi 1) , YANG Bo 2) ,GUAN Fangxia 3) , DU Ying 4) , SONG laijun 2) ZHANG Qingyong 5) , HU Xiang 6) , HU Pingsheng 7) , XU Hong 8) 1)Graduate of Department of Neurosurgery, Zhengzhou University, Zhengzhou 450052 2)Department of Neurosurgery,the First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052 3)Visiting Professor,Department of Biomedicine and Bioengineering, Zhengzhou University, Zhengzhou 450052 4)Department of Microbiology and Immunology, College of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450052 5)Department of Thoracic Surgery,the Second Affiliated Hospital, Zhengzhou University, Zhengzhou 450014 6)Shenzhen Beike Cell Engineering Institute,Shenzhen 518000 7)Biovitron Company,Sweden 999027 8)Henan Institute of Tumor Therapy and Prevention, Zhengzhou 450008

【机构】 郑州大学神经外科学； 郑州大学第一附属医院神经外科； 郑州大学生物医学工程学； 郑州大学基础医学院微生物与免疫学教研室； 郑州大学第二附属医院胸外科； 深圳市北科细胞工程研究所； 瑞典Biovitron公司； 河南省肿瘤研究所 硕士研究生郑州450052； 郑州450052； 特聘教授郑州450052； 郑州450014； 深圳518000； 999027； 郑州450008；

【摘要】 目的探讨来源于人羊膜间质干细胞(amniotic-derivedmesenchymalstemcells,AD-MSCs)的分离、培养及鉴定。方法无菌条件下取正常足月剖腹产胎儿的羊膜剪成碎片,加入胰酶37℃消化30min,共2次,然后加入终浓度1.0g/L的胶原酶和0.1g/L的DNA酶,37℃消化60min,收集细胞,用含体积分数为10%胎牛血清(FBS)的DMEM/F12培养基进行培养和纯化。倒置显微镜观察细胞形态,取4~6代的细胞用流式细胞仪分析细胞表面标志,取扩增第3代后的AD-MSCs加入全反式维甲酸(RTRA)和碱性成纤维细胞生长因子(bFGF)向神经元样细胞诱导分化。结果来源于羊膜的细胞种植于DMEM/F12培养基中后,可在体外大量扩增并纯化;流式细胞检测显示CD29、CD44、HLA-ABC阳性表达,CD34、CD45、HLA-DR阴性表达。免疫细胞化学显示AD-MSCs经RTRA和bFGF诱导后表达神经元特异性烯醇化酶,不表达胶质纤维性蛋白。结论羊膜中存在间质干细胞,其可以在体外培养、扩增、纯化,并可在体外向神经元样细胞分化。

【Abstract】 Aim: To investigate the isolation, culture and identification of human amniotic-derived mesenchymal stem cells(AD-MSCs). Methods:The amniotic membrane was collected from full term cesarean section. The harvested pieces of tissue were mechanically minced and treated with trypsin twice at 37°C for 30 min. Then the tissue pieces were placed in DMEM/F12 containing collagenase (1.0 g/L) and DNase (0. 1 g/L) and were incubated at 37°C for 60 min. The cells were cultured and purified with complete medium consisting of DMEM/F12 and 10% fetal bovine serum. The morphological changes of the cells were observed under phase contrast microscope, and the cell surface antigenic characteristics of the cultured AD-MSCs at passage 4~6 were analyzed by flow cytometry. The differentiation of AD-MSCs were induced by RTRA and bFGF. Results: The AD-MSCs could proliferate in vitro. The flow cytometry analyses revealed that the expression of surface antigens, such as CD29, CD44 and HLA-A,B,C(MHC class I) was positive; but CD34, CD45, and HLA-DR(MHC class Ⅱ)were negative. After induced by RTRA and bFGF,AD-MSCs expressed the specific markers of neurons (NSE) by immunocytochemistry. Conclusion: The amniotic membrane contains stem cells that are capable of rapid expansion and differentiation into neuron-like-cells in vitro.

【关键词】 羊膜； 间质干细胞； 培养； 神经元样细胞；
【Key words】 amniotic membrane； mesenchymal stem cell； culture； neuron-like cell；
【基金】 河南省医学科技创新人才工程项目2005018
  • 【文献出处】 郑州大学学报(医学版) ,Journal of Zhengzhou University(Medical Sciences) , 编辑部邮箱 ,2006年02期
  • 【分类号】R329
  • 【被引频次】34
  • 【下载频次】370
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