中国现代神经疾病杂志 ›› 2014, Vol. 14 ›› Issue (11): 1000-1006. doi: 10.3969/j.issn.1672-6731.2014.11.015

• 基础研究 • 上一篇    下一篇

2 体外构建纯化骨髓间充质干细胞与壳聚糖生物支架复合体的实验研究

闫峰, 张越林, 岳伟, 毛国超, 王刚, 左振兴, 高珂   

  1. 710068 陕西省人民医院神经外科(闫峰,张越林,毛国超,王刚);300060 天津市环湖医院神经内科(岳伟);710061 西安交通大学第一附属医院神经外科(左振兴,高珂)
  • 出版日期:2014-11-25 发布日期:2014-11-29
  • 通讯作者: 张越林 (Email:zhangyuelin68@163.com)
  • 基金资助:

    陕西省科技研究发展计划项目(项目编号:2012KCT-16)

Experimental study on cultivation and purification of bone marrow-derived mesenchymal stem cells and its co-culture with chitosan porous scaffolds in vitro

YAN Feng1, ZHANG Yue-lin1, YUE Wei2, MAO Guo-chao1, WANG Gang1, ZUO Zhen-xing3, GAO Ke3   

  1. 1Department of Neurosurgery, Shanxi Provincial People's Hospital, Xi'an 710068, Shanxi, China
    2Department of Neurology, Tianjin Huanhu Hospital, Tianjin 300060, China
    3Department of Neurosurgery, the First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, Shanxi, China
  • Online:2014-11-25 Published:2014-11-29
  • Contact: ZHANG Yue-lin (Email: zhangyuelin68@163.com)
  • Supported by:

    This study was supported by Science and Technology Research and Development Program of Shanxi Province (No. 2012KCT-16).

摘要: 研究背景 壳聚糖作为组织工程中的常用支架材料,具有生物降解性强、抗原性低、生物相容性佳和无热原反应等优势。尝试将经分离纯化的SD 大鼠骨髓间充质干细胞与自制的可吸收壳聚糖多孔支架共培养,研究其生物相容性,以为今后应用细胞-支架复合体植入中枢神经系统行替代治疗,实现神经细胞再生、神经功能修复奠定基础。方法 采用全骨髓贴壁培养法分离3 周龄SD 大鼠骨髓间充质干细胞,选择第3 代细胞行流式细胞术纯化鉴定并与壳聚糖多孔支架于体外三维立体环境共培养。乙醇替代法检测壳聚糖多孔支架孔隙率;扫描电子显微镜观察支架内部结构、测量孔径大小,以及细胞在支架内部生长状态及其与生物支架融合情况;MTT 法测定细胞在支架内增殖状况。结果 经体外培养的大鼠骨髓间充质干细胞形态均匀、呈纤维状排列,混杂细胞比例明显减少;CD29 和CD45RA 阳性表达率分别为98.49%和0.85%,达高纯度表达;支架孔隙率为90%,支架内部为孔径均匀、相通的三维立体结构,细胞紧密贴附于支架微孔内壁上、黏附牢固,可见伪足伸出,与支架融合较为理想。支架对骨髓间充质干细胞增殖无明显影响,细胞生长状况良好。结论 壳聚糖多孔支架具备良好的孔径和孔隙率等性状,与骨髓间充质干细胞生物相容性良好,可作为干细胞移植的支架载体为今后细胞替代治疗中枢神经系统疾病的转化医学研究奠定基础。

关键词: 骨髓细胞, 间质干细胞, 壳聚糖, 生物相容性材料, 细胞, 培养的, 流式细胞术, 显微镜检查, 电子, 扫描

Abstract: Background  As commonly used scaffold material in tissue engineering, chitosan has many advantages, such as strong biodegradability, low antigenicity, good biocompatibility and no pyrogen reaction. This study aims to isolate, cultivate and purify Sprague-Dawley (SD) rat bone marrow-derived mesenchymal stem cells (BMSCs), and to observe the growth of BMSCs when co-cultured with self-made chitosan porous scaffold in vitro and to test the biocompatibility of this tissue engineering scaffold, so as to lay the foundation for promoting nerve regeneration of transplant treatment.  Methods  Three-week-old healthy male SD rats were used in this study, and BMSCs were isolated and purified through bone marrow adherent culture method. The surface markers of BMSCs at Passage 3 were detected and identified by flow cytometry (FCM) and the BMSCs were three?dimensionally cultured in vitro on chitosan porous scaffolds produced by freeze-drying method. Ethanol alternative method was used to detect the chitosan scaffold porosity. Scanning electron microscope was used to explore the internal structure of the scaffold, measure the size of its aperture, and observe the morphology and development of the cells within the scaffold. Methyl thiazolyl tetrazolium (MTT) method was used to determine the cells' proliferation.  Results  The cultured BMSCs were uniform and similiar to fibrous arrangement, and mixed cells reduced obviously. The identification result of FCM showed the CD29 positive rate was 98.49% and CD45RA positive rate was only 0.85%. The chitosan scaffold had an interlinked, uniform similar three-dimensional porous structure and its aperture porosity was 90%. Some cells stretched out pseudopod and infiltrated into the porous structure of scaffold, even fusing with them. The BMSCs were seeded in the scaffold successfully. The chitosan scaffold had no obvious effect on BMSCs' proliferation. Conclusions  Chitosan porous scaffolds have good structural character and biocompatibility, and can be used as an alternative cell-carrier for later cell replace treatment of central nervous system diseases.

Key words: Bone marrow cells, Mesenchymal stem cells, Chitosan, Biocompatible materials, Cells, cultured, Flow cytometry, Microscopy, electron, scanning