中国组织工程研究

中国组织工程研究 ›› 2018, Vol. 22 ›› Issue (13): 1993-1998.doi: 10.3969/j.issn.2095-4344.0427

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

骨髓间充质干细胞在Matrigel凝胶支架上的生长与变化

张斌斌1，高全文2，李 冰3，黄 沙4，姚 斌4   

  1. 1山西医科大学口腔医学系，山西省太原市 030001；2解放军总医院第一附属医院烧伤整形科，北京市 100048；3山西医科大学口腔医院，山西省太原市 030001；4解放军总医院基础医学研究所，北京市  100853
  • 修回日期:2017-11-21 出版日期:2018-05-08 发布日期:2018-05-08
  • 通讯作者: 李冰，博士，副院长，主任医师，硕士生导师，山西医科大学口腔医院，山西省太原市 030001; 并列通讯作者：高全文，博士，副主任医师，解放军总医院第一附属医院烧伤整形科，北京市 100048
  • 作者简介:张斌斌，男，1990年生，山西省太原市人，汉族，山西医科大学2015级硕士研究生，主要从事口腔颌面外科及修复的基础研究。
  • 基金资助:

    北京市自然科学基金青年基金项目(7144249)

Bone marrow mesenchymal stem cells on Matrigel: growing and changing

Zhang Bin-bin1, Gao Quan-wen2, Li Bing3, Huang Sha4, Yao Bin4   

  1. 1Department of Stomatology, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China; 2Department of Burns and Plastic Surgery, First Hospital Affiliated to General Hospital of PLA, Beijing 100048, China; 3Hospital of Stomatology, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China; 4Institute of Basic Medical Sciences, General Hospital of PLA, Beijing 100853, China
  • Revised:2017-11-21 Online:2018-05-08 Published:2018-05-08
  • Contact: Li Bing, M.D., Chief physician, Master’s supervisor, Hospital of Stomatology, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China; Gao Quan-wen, M.D., Associate chief physician, Department of Burns and Plastic Surgery, First Hospital Affiliated to General Hospital of PLA, Beijing 100048, China
  • About author:Zhang Bin-bin, Studying for master’s degree, Department of Stomatology, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
  • Supported by:

    the National Natural Science Foundation of Beijing, No. 7144249

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摘要：

文章快速阅读：

文题释义：
三维细胞培养技术：是指将具有三维结构的不同材料载体与各种不同种类的细胞在体外共同培养，使细胞能够在三维立体空间结构中迁移、生长，构成三维的细胞-载体复合物。三维细胞培养是介于单层细胞培养与动物实验之间的一种技术，既能最大程度的模拟体内环境，又能展现细胞培养的直观性及条件可控性的优势。
Matrigel：是从富含胞外基质蛋白的EHS小鼠肿瘤中提取出基底膜基质，其主要成分有层粘连蛋白、Ⅳ型胶原、巢蛋白、硫酸肝素糖蛋白，还包含生长因子和基质金属蛋白酶等。在室温条件下，Matrigel聚合形成具有生物学活性的三维基质，模拟体内细胞基底膜的结构、组成、物理特性和功能，有利于体外细胞的培养和分化，可用于对细胞形态、生化功能、迁移、侵染和基因表达等研究。

 

摘要
背景：研究表明，Matrigel作为一种细胞外基质复合物加入到细胞培养体系中，对细胞的增殖、分化、胶原分泌具有促进作用。
目的：建立Matrigel复合骨髓间充质干细胞的三维培养模型，观察Matrigel三维培养条件下骨髓间充质干细胞的形态、增殖及存活情况。
方法：全骨髓贴壁法分离培养骨髓间充质干细胞，进行成骨、成脂诱导鉴定；CCK-8测定第3代骨髓间充质干细胞的生长曲线；选取生长状态良好的第2代骨髓间充质干细胞，与Matrigel混合形成细胞凝胶复合物，采用相差显微镜和苏木精-伊红染色观察骨髓间充质干细胞的形态及增殖情况，Live/Dead染色评价细胞活性。
结果与结论：①全骨髓贴壁法分离培养的比格犬骨髓间充质干细胞具备向成骨、成脂细胞分化的能力；②骨髓间充质干细胞的生长曲线呈S形，符合正常细胞的生长特性；③相差显微镜下可见骨髓间充质干细胞在Matrigel中逐渐伸长相互交联，呈三维网状生长，增殖状态良好，随着时间延长(7 d后)，基质胶逐渐变软塌陷，14 d时仍可见少部分细胞网状交联生长；苏木精-伊红染色可见第4天时细胞胞质较大，第7天时细胞细长、相互交联；④三维培养第1，4，7天，骨髓间充质干细胞在Matrigel中活性百分比分别为92.57%，95.54%，97.37%，呈逐渐升高趋势；⑤结果表明，骨髓间充质干细胞在Matrigel中的增殖能力较强，活性较高。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
ORCID: 0000-0002-0811-2889(张斌斌)

关键词: 干细胞, 骨髓间充质干细胞, 三维培养, Matrigel, 细胞增殖, 细胞活性, 北京市自然科学基金

Abstract:

BACKGROUND: Matrigel as an extracellular matrix complex can facilitate cell proliferation, differentiation and collagen secretion in a cell culture system.
OBJECTIVE: To establish a three-dimensional culture model of Matrigel combined with bone marrow mesenchymal stem cells (BMSCs) and to observe the morphology, proliferation and survival of BMSCs in the Matrigel three-dimensional culture model.
METHODS: BMSCs were isolated and cultured by the whole bone marrow adherent method, followed by osteogenic and adipogenic induction and identification. The growth curve of passage 3 BMSCs was determined through the CCK-8 experiment. Passage 2 BMSCs were combined with Matrigel, and the morphology and proliferation of BMSCs were observed by hematoxylin-eosin staining under a phase contrast microscope. The viability of the cells was evaluated by the Live/Dead staining.
RESULTS AND CONCLUSION: (1) BMSCs cultured by the whole bone marrow adherent method had the ability to differentiate into osteoblasts and adipocytes. (2) BMSCs exhibited an “S”-shaped growth curve, which was consistent with the growth characteristics of normal cells. (3) Under the phase contrast microscope, BMSCs cultured by the Matrigel model were extended and interconnected in a three-dimensional network growth state with good proliferation. The Matrigel gradually became soft and collapsed over time (7days) and a few cells were still in a network growth after 14 days. Hematoxylin-eosin staining results showed that the cell cytoplasm was larger at 4 days and the cells became thinned and mutually cross-linked at 7 days. (4) The active percentage of BMSCs in the Matrigel model was 92.57%, 95.54% and 97.37% at 1, 4, 7 days of culture, respectively. To conclude, BMSCs cultured on the Matrigel has good proliferation and high viability. 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Bone Marrow, Mesenchymal Stem Cells, Extracellular Matrix, Cell Proliferation, Tissue Engineering

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