中国组织工程研究

中国组织工程研究

• 干细胞转基因表达 • 上一篇    下一篇

利用基因芯片分析碱性成纤维细胞生长因子诱导脐血干细胞CD34+与CD133+细胞基因表达的差异

陈慧萍1，李倩如2，张 静1，杜 英2，杨 波3，李国喜4，胡 祥4，董子明2   

  1. 1郑州大学医学院07级在读硕士，河南省郑州市  450001；
    2郑州大学基础医学院微生物学与免疫学教研室，河南省郑州市  450001;
    3郑州大学第一附属医院神经外科，河南省郑州市  450052；
    4深圳市北科细胞工程研究所，广东省深圳市  518057
  • 出版日期:2010-01-04 发布日期:2010-01-04
  • 通讯作者: 杜英，博士，教授，硕士生导师，郑州大学基础医学院微生物学与免疫学教研室，河南省郑州市 450001 duying@zzu.edu.cn
  • 作者简介:陈慧萍，女，1978年生，河南省新郑市人，汉族，郑州大学医学院在读硕士，主要从事干细胞生物学特性及其应用的研究。 huiping918@yahoo.com.cn
  • 基金资助:

    广东省科技计划项目(2007B031500015)。

Basic fibroblast growth factor-induced differences in gene expression of human umbilical cord blood CD34+ and CD133+ stem cells: Gene chip analysis

Chen Hui-ping1, Li Qian-ru2, Zhang Jing1, Du Ying2,Yang Bo3, Li Guo-xi4, Hu Xiang4, Dong Zi-ming2   

  1. 1Master 07, Medical College of Zhengzhou University, Zhengzhou  450001, Henan Province, China;
    2Department of Microbiology and Immunology, College of Basic Medical Sciences, Zhengzhou University, Zhengzhou   450001, Henan Province, China;
    3Department of Neurosurgery, First Affiliated Hospital, Zhengzhou University, Zhengzhou   450052, Henan Province, China; 4Shenzhen Beike Cell Engineering Institute, Shenzhen   518057, Guangdong Province, China
  • Online:2010-01-04 Published:2010-01-04
  • Contact: Du Ying, Doctor, Professor, Master’s Supervisor, Department of Microbiology and Immunology, College of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450001, Henan Province, China duying@zzu.edu.cn
  • About author:Chen Hui-ping, Studying for master’s degree, Master 07, Medical College of Zhengzhou University, Zhengzhou 450001, Henan Province, China huiping918@yahoo.com.cn
  • Supported by:

    the Scientific Research Project of Guangdong Province, No. 2007B031500015*

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摘要：

背景：迄今为止，人们利用基因芯片对碱性成纤维细胞生长因子诱导CD34+和CD133+干细胞分化后细胞生物学性状、功能调控、基因变化及其表达差异等方面的认识尚不足，有待深入研究。

目的：利用基因芯片比较脐血CD34+和CD133+细胞基因表达的差异，并探讨碱性成纤维细胞生长因子体外诱导脐血CD34+和CD133+造血干/祖细胞分化的基因表达变化，及其对碱性成纤维细胞生长因子反应性差异。

方法：利用MiniMACS免疫磁珠法从脐静脉血单个核细胞中分离出CD34+和CD133+干/祖细胞，经含碱性成纤维细胞生长因子、B27的DMEM/F12细胞营养液培养10~15 d，提取培养前后细胞总RNA，利用Oligo GEArray®芯片和GEArray表达分析软件进行芯片数据分析。流式细胞仪检测CD34+和CD133+造血干细胞回收率。碱性成纤维细胞生长因子诱导前后CD34+、CD133+细胞形态变化。变性琼脂糖凝胶电泳测定RNA浓度和纯度。基因芯片检测结果。

结果与结论：①对20份脐血分别进行CD34+和CD133+细胞分选，分选CD34+细胞纯度为(77.52±5.06)%，回收率为(2.74±1.59)%；CD133+细胞纯度为(79.16±3.37)%，回收率为(1.12±0.94)%。②新分选出的CD34+细胞呈球形，经碱性成纤维细胞生长因子培养15 d后，多数细胞形态未发现显著变化，部分细胞出现贴壁生长，可见突起和梭形细胞。CD133+细胞呈球形，经碱性成纤维细胞生长因子培养15 d后，细胞明显扩增，由圆球形变为不规则形，部分细胞出现贴壁生长。③CD34+干细胞培养前后总RNA提取质量分别为2 236 ng和1 796 ng；CD133+干细胞培养前后总RNA提取质量分别为2 518 ng和2 191 ng。④在所检测的263个干细胞相关基因中，脐血CD133+细胞与CD34+细胞基因表达差异2倍以上的基因有10种，其中5种基因表达前者高于后者，5种基因表达前者低于后者；碱性成纤维细胞生长因子诱导培养后，CD133+细胞有32种基因表达显著高于CD34+细胞，在检测的263个基因中，未见低于CD34+细胞的基因。证实脐血中新分离的CD133+细胞和CD34+细胞基因表达仅有少数基因存在差异；CD133+细胞对碱性成纤维细胞生长因子的反应性显著强于CD34+细胞，表现为细胞周期调节、信号转导和分化等基因表达增强。

关键词: 碱性成纤维细胞生长因子, CD34, CD133, 基因芯片, 脐血

Abstract:

BACKGROUND: Further studies are needed to understand the cytobiological character, functional regulation, gene changes and expression difference of CD34+ and CD133+ stem cells induced by basic fibroblast growth factor (bFGF) using gene chip.

OBJECTIVE: To compare the differences of gene expression and the response to bFGF of human umbilical cord CD34+ and CD133+ cells, and to explore gene expression changes of bFGF-induced umbilical cord CD34+ and CD133+ hematopotic stem cells/hemapoietic progenitor cells in vitro.

METHODS: Human umbilical cord blood CD34+ and CD133+ cells were isolated and purified by MiniMACS immunomagnetic beads selection. The CD34+ and CD133+cells were cultured for 10 to 15 days in DMEM/F12 medium, supplemented with bFGF and B27. Total RNA from these cells was extracted and the genetic level of these cells was performed using Oligo GEArray® chip and GEArray software. Selected rate of CD34+ and CD133+ hematopoietic stem cells was detected using flow cytometry. CD34+ and CD133+ cell morphological changes were measured before and after bFGF induction. The concentration and purity of RNA were determined by agarose gel electrophoresis degeneration. Gene-chip test results were analyzed.

RESULTS AND CONCLUSION: ①The 20 samples of cord blood were isolated and purified respectively, CD34+ cell purity (77.52±5.06)%, recovery rate (2.74±1.59)%; CD133+ cell purity (79.16±3.37) % and the recovery (1.12±0.94)%. ②The new selected CD34+ cells were spherical. Following induced by bFGF for 15 days, the majority of cell morphology did not find significant changes, some cells were adherent growth, and protruding and spindle cells were seen. CD133+ cells were spherical, by bFGF in cultured 15 days later, cells were significantly amplified, the round shape changed into an irregular shape, and some cells were adherent growth. ③The total RNA of CD34+ stem cells before and after incubation was respectively 2 236 ng and 1 796 ng. The total RNA of CD133+ stem cells before and after induction was respectively 2 518 ng and 2 191 ng. ④In the detection of 263 genes related to stem cells, two-fold differences of 10 genes in umbilical cord blood CD34+ cells and CD133+ cells, including five kinds of genes expression were higher in the former than the latter, five kinds of genes expression were lower in the former than the latter. After bFGF-induced culture, 32 kinds of gene expression of CD133+ cells were significantly higher than CD34+ cells. Among detected 263 genes, no gene was lower than CD34+ cells. There were only a few gene expression differences of fresh-separated cord blood CD133+ cells and CD34+ cells. The response of CD133+ cells to bFGF was significantly stronger than CD34+ cells, which manifested cell cycle regulation, signal transduction and differentiation, gene expression enhanced.

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