中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (10): 1721-1728.doi: 10.3969/j.issn.1673-8225.2010.10.003

• 干细胞培养与分化 • 上一篇    下一篇

人脐带沃顿胶间充质干细胞与脑肿瘤干细胞的共培养

田 毅1,2，关方霞3，胡 祥4,5，杨 波1,2，杜 英6，周长辉3，巴云涛1，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；郑州大学，
    3生物工程系，7临床医学系，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；
    8湖南师范大学生命科学学院，湖南省长沙市  410081
  • 出版日期:2010-03-05 发布日期:2010-03-05
  • 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:田 毅，男，1982年生，河南省焦作市人，汉族，郑州大学医学院在读硕士，医师，主要从事间充质干细胞和脑肿瘤干细胞方面的研究。ethan43@qq.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研究。guanfangxia@126.com
  • 基金资助:

    郑州大学211三期建设项目“干细胞基础与临床研究”；江苏省干细胞与生物治疗公共技术服务平台发展(BM2008146)项目。

Coculture of human umbilical cord mesenchymal stem cells from Wharton’s jelly and brain tumor stem cells

Tian Yi1,2, Guan Fang-xia3, Hu Xiang4,5, Yang Bo1,2, Du Ying6, Zhou Chang-hui3, Ba Yun-tao1, Gu Chen-xi7, Lei Ning-jing3, Wang Xiao-wei8   

  1. 1Department of Neurosurgery, First Affiliated Hospital, Zhengzhou University, Zhengzhou   450052, Henan Province, China;
    2Institute of Clinical Medicine, Zhengzhou University, Zhengzhou   450052, Henan Province, China;
    3Department of Bioengineering, 7Department of Clinical Medicine, 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;
    8College of Life Science, Hunan Normal University, Changsha   410081, Hunan Province, China
  • Online:2010-03-05 Published:2010-03-05
  • Contact: Yang Bo, Master, Professor, Doctoral supervisor, Department of Neurosurgery, First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, Henan Province, China; Institute of Clinical Medicine, Zhengzhou University, Zhengzhou 450052, Henan Province, China yangbo96@126.com
  • About author:Tian Yi, Studying for Master’s degree, Physician, Department of Neurosurgery, First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, Henan Province, China; Institute of Clinical Medicine, Zhengzhou University, Zhengzhou 450052, Henan Province, China ethan43@qq.com Guan Fang-xia, Doctor, Professor, Department of Bioengineering, Zhengzhou University, Zhengzhou 450001, Henan Province, China guanfangxia@126.com Tian Yi and Guan Fang-xia contributed equally to this article.
  • Supported by:

    the Zhengzhou University “211 Project-Phase Ⅲ”- the Basic and Clinical Research of Stem Cells*; Jiangsu Public Technology Service Platform Development Program of Stem Cells and Biotherapy, No. BM2008146*

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摘要：

背景：人脐带沃顿胶间充质干细胞满足了国际细胞治疗协会规定的间充质干细胞的特点，能够向骨、软骨、脂肪、肌肉、神经细胞诱导分化并支持其他干细胞的扩增，对免疫系统有良好的耐受性，对肿瘤有定向迁徙性。
目的：观察人脐带沃顿胶间充质干细胞与脑肿瘤干细胞体外共培养后，脑肿瘤干细胞的生物学变化。
方法：以原位法培养人脐带沃顿胶间充质干细胞，以酶消化法培养人脑肿瘤组织脑肿瘤干细胞，以细胞传代法获取第3代细胞。应用不添加任何生长因子的无血清培养基将两种细胞在24孔板中进行直接共培养。第3，7天应用流式细胞术检测细胞CD133表达；应用免疫荧光法检测贴壁细胞巢蛋白和胶质纤维酸性蛋白表达；将第3天离心所得的共培养上清液重悬第3代脑肿瘤干细胞并与正常培养悬浮的第3代脑肿瘤干细胞置入96孔板中，应用酶标仪检测两组细胞生长曲线的差异。
结果与结论：两种细胞共培养后在倒置显微镜下可见脑肿瘤干细胞球随着培养时间的增加出现分解、贴壁、分化现象；贴壁的脑肿瘤干细胞免疫荧光染色胶质纤维酸性蛋白和巢蛋白均阳性。恶性程度高的脑肿瘤组织培养的脑肿瘤干细胞表达CD133量越高，而与沃顿胶间充质干细胞共培养后随着时间的变化均出现CD133表达量降低。共培养3 d的上清液培养的脑肿瘤干细胞与正常培养基培养的脑肿瘤干细胞相比，增殖明显受到抑制。结果显示沃顿胶间充质干细胞与脑肿瘤干细胞体外共培养后可限制脑肿瘤干细胞表面标记物CD133的阳性率以及细胞增殖能力并促使其分化。

关键词: 共培养, 脑肿瘤干细胞, CD133, 沃顿胶间充质干细胞, 人脐带

Abstract:

BACKGROUND: Human mesenchymal stem cells derived from Wharton’s jelly (WJCs) display the characteristics of MSCs as defined by the International Society for Cellular Therapy. They can be differentiated into bone, cartilage, adipose, muscle, and neural cells. They can also support the expansion of other stem cells, be well-tolerated by the immune system, and have the ability to home to tumors.

OBJECTIVE: To investigate biological changes of WJCs and brain tumor stem cells (BTSCs) co-cultured in vitro.

METHODS: WJCs cultured by situ cultivation and BTSCs used enzyme digestion way respectively, and gathering the 3rd passage of WJCs though subculturing as well as BTSCs. Two kinds of cells co-cultured in 24-well plates in serum-free medium (SFM) without any growth factor. 3 and 7 days after co-cultured respectively, CD133 expression of suspension cells in the 24-well plates were identified by flow cytometry, and immunofluorescence was performed for Nestin and glial fibrillary acidic protein (GFAP) expression of adherent cells. Co-culture supernatant (CCS) re-suspended 3rd passage of BTSCs and cultured into 96-well plates at day 3, which were used to determine the difference in cell growth curve in both groups using a microplate reader.

RESULTS AND CONCLUSION: With the cocultivation days increasing, the phenomenon that tumor sphere cells began to be decomposed, adherent and differentiated observed by an inverted microscope. BTSCs in the co-cultured group expressed GFAP and Nestin when adherent and differentiated. The higher degree of malignant brain tumor tissue used in culturing BTSCs was, the higher expression of CD133 in BTSCs was. CD133+ in BTSCs declined when co-cultured with WJCs. Growth curve of brain tumor stem cells cultured in CCS compared with in SFM at day 3, which indicates that the proliferation of BTSCs inhibited obviously. Results indicated that CD133+ expression and proliferative capacity of BTSCs went down and BTSCs underwent differentiation during the co-culture in vitro.

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