中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (19): 3513-3517.doi: 10.3969/j.issn.1673-8225.2010.19.019

• 干细胞因子及调控因子 stem cell factors and regulatory factors • 上一篇    下一篇

人脐带Wharton’s jelly源间充质干细胞培养中碱性成纤维细胞生长因子的作用

巴云涛1,2，关方霞3，胡 祥4,5，杨 波1,2，杜 英6，张天祥1，田 毅1，乔晓俊1，王成春3，谷晨熙7，雷宁静3，王晓薇8   

  1. 1郑州大学第一附属医院神经外科，河南省郑州市 450052；
    2河南省高等学校临床医学重点学科开放实验室，河南省郑州市  450052； 
    3郑州大学生物工程系，河南省郑州市  450001；
    4江苏省干细胞与生物治疗公共技术服务平台，江苏省泰州市 225300；
    5深圳市北科细胞工程研究所，广东省深圳市 518000；
    6郑州大学基础医学院微生物与免疫学教研室，河南省郑州市  450052；
    7郑州大学临床医学系，河南省郑州市 450001；
    8湖南师范大学生命科学学院，湖南省长沙市  410081
  • 出版日期:2010-05-07 发布日期:2010-05-07
  • 通讯作者: 杨 波，硕士，教授，博士生导师，郑州大学第一附属医院神经外科，河南省郑州市 450052 yangbo96@126.com
  • 作者简介:巴云涛，男，1979年生，河南省郸城县人，汉族，郑州大学医学院在读硕士，主治医师，主要从事间充质干细胞和脑肿瘤干细胞方面的研究。 bayuntao790421@126.com 并列第一作者：关方霞，女，1969年生，陕西省西安市人，汉族，1999年山东医科大学毕业，博士，教授，主要从事间质干细胞与肿瘤干细胞方面的研究。 guanfangxia@126.com

Effects of basic fibroblast growth factor in culture of mesenchymal stem cells derived from Wharton’s jelly of human umbilical cord

Ba Yun-tao1,2, Guan Fang-xia3, Hu Xiang4,5, Yang Bo1,2, Du Ying6, Zhang Tian-xiang1, Tian Yi1, Qiao Xiao-jun1, Wang Cheng-chun3, Gu Chen-xi7, Lei Ning-jing3, Wang Xiao-wei8   

  1. 1Department of Neurosurgery, First Affiliated Hospital, Zhengzhou University, Zhengzhou  450052, Henan Province, China;
    2Opening Laboratory for Key Subject, Henan Institute of Clinical Medicine, Zhengzhou  450052, Henan Province, China;
    3Department of Bioengineering, Zhengzhou University, Zhengzhou  450001, Henan Province, China;
    4Jiangsu Public Technology Service Platform of Stem Cells and Biotherapy, Taizhou  225300, Jiangsu Province, China;
    5Shenzhen Beike Cell Engineering Institute, Shenzhen  518000, Guangdong Province, China;
    6Department of Microbiology and Immunology, College of Basic Medical Sciences, Zhengzhou University, Zhengzhou  450052, Henan Province, China;
    7Department of Clinical Medicine, Zhengzhou University, Zhengzhou  450001, Henan Province, China;
    8College of Life Science, Hunan Normal University, Changsha  410081, Hunan Province, China
  • Online:2010-05-07 Published:2010-05-07
  • Contact: Yang Bo, Master, Professor, Doctoral supervisor, Department of Neurosurgery, First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, Henan Province, China; Opening Laboratory for Key Subject, Henan Institute of Clinical Medicine, Zhengzhou 450052, Henan Province, China yangbo96@126.com
  • About author:Ba Yun-tao, Studying for master’s degree, Attending physician, Department of Neurosurgery, First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, Henan Province, China; Opening Laboratory for Key Subject, Henan Institute of Clinical Medicine, Zhengzhou 450052, Henan Province, China bayuntao790421@126.com Guan Fang-xia, Doctor, Professor, Department of Bioengineering, Zhengzhou University, Zhengzhou 450001, Henan Province, China guanfangxia@126.com Ba Yun-tao and Guan Fang-xia contributed equally to this article.
  • Supported by:

    Third-Stage Construction Project of 211 Program in Zhengzhou University*;
    the Jiangsu Taizhou Public Technology Service Platform of Medicine, No. BM2008146*

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摘要：

背景：培养脐带源间充质干细胞过程中，细胞形态常易变得宽大不规则，传代不易贴壁，死亡率高，找到一个维持细胞稳定的方法是有必要的。

目的：拟采用组织块培养法从人脐带Wharton’s jelly中分离培养间充质干细胞，观察碱性成纤维细胞生长因子对其生物学特性的影响。

方法：采用组织块法分离培养和扩增脐带组织间充质干细胞，对照组用DMEM/F12 培养基+体积分数为10%胎牛血清培养，实验组用DMEM/F12 培养基+体积分数为10%胎牛血清+ 20 μg/L碱性成纤维细胞生长因子培养。观察组织块游出细胞的时间和细胞形态，每隔3 d或4 d更换培养液，待细胞达到80%~90%融合时，用0.25%胰酶消化后，按1∶2或1∶3的比例传代。

结果与结论：倒置显微镜下观察对照组中从8~10 d开始有细胞从组织块四周游出贴壁，实验组中从6~8 d开始有细胞从组织块四周游出贴壁，1周后可见多数呈长梭形或扁平形的成纤维样细胞，围绕组织块成旋涡状，前3代细胞形态及传代间隔时间两组基本相同；3代以后实验组细胞较对照组易于贴壁，传代死亡率低，生长曲线提示增殖能力强。流式细胞术分析两组细胞周期显示第3，6代 70%以上的细胞处于G0/G1期，且第3，6代细胞均阳性表达CD29，CD44，弱表达HLA-ABC，不表达CD34、CD45和 HLA-DR。结果提示采用组织块培养法可从人脐带Wharton’s jelly中分离出具有间充质干细胞特性的细胞，且碱性成纤维细胞生长因子能使细胞从组织块游出时间提前和有助于促使细胞贴壁，一定程度上维持细胞形态稳定性，提高增殖能力，并且不改变细胞的表面标志物表达。

关键词: 碱性成纤维细胞生长因子, 间充质干细胞, 人脐带, Wharton&rsquo, s jelly, 干细胞

Abstract:

BACKGROUND: During culture of mesenchymal stem cells (MSCs) derived from Wharton’s jelly of human umbilical cord. MSC morphology tends to become hypertrophic and irregular. MSCs were found to have higher rate of death and not be adapt to be adherent after passage. It is necessary to find a method to maintain its stability.

OBJECTIVE: To isolate MSCs from human umbilical cord wharton’s jelly by tissue block method, and to investigate basic fibroblast growth factor (bFGF) effects on biological characteristics of MSCs.

METHODS: Human umbilical cord mesenchymal stem cells (hUCMSCs) were separated by tissue block method. Tissue fragments in control group were cultured in growth medium consisting of Dulbecco’s Modified Essential /F12 Media (DMEM/F12) and 10% volume fraction of fetal bovine serum. Tissue fragments in experiment group were cultured in growth medium including 20 μg/L bFGF besides these in control group. Time of tissue blocks emigrating from cells and morphology of cells were observed. The medium was changed every 3-4 days. When the cells reached 80%-90% confluency, they were detached with 0.25% trypsin and were passaged at a ratio of 1: 2 or 1:3.

RESULTS AND CONCLUSION: A few long spindle or flat fibroblast-like cells were presented firstly after 8-10 days of incubation in control group, while in experiment group it took 6-8 days. One week later, lots of long spindle or flat fibroblast-like cells like whirlpool around micro-mass were presented in two groups. In the first 3 passages, cell morphous were similar and cells were passaged at approximately equivalent time. After passage 3, cells in experiment group were easier to be adherent, lower rate of death and better proliferation ability (shown by growth curve) in comparison with these in control group. Flow cytometry revealed that cell cycle at the passages 3 and 6 showed the percentage of G0/G1 was more than 70% respectively. CD44 and CD29 were highly expressed on the surface of passages 3 and 6 cells, whereas the expression of HLA-ABC was less positive, but there was negative for CD34, CD45 and HLA-DR. Cells from Wharton’s jelly of the human umbilical cords, which have the biological characteristics of MSCs, have been isolated by tissue culture method. bFGF can shorten the time that MSCs are presented firstly, improve its adherence and proliferation ability and maintain its morphological stability in some degree, moreover keep surface marker expression stability of MSCs.

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