中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (14): 2497-2502.doi: 10.3969/j.issn.1673-8225.2010.14.006

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

不同血清微环境对大鼠骨髓间充质干细胞体外培养的影响

郑景辉，李勇华，王丽萍，简维雄，黄献平，袁肇凯   

  1. 湖南中医药大学中医诊断研究所，湖南省长沙市  410007
  • 出版日期:2010-04-02 发布日期:2010-04-02
  • 通讯作者: 袁肇凯，教授，湖南中医药大学中医诊断研究所，湖南省长沙市 410007 yuanzk1520@yahoo.com.cn
  • 作者简介:郑景辉☆，男，1976年生，山东省阳信县人，汉族，湖南中医药大学在读博士，中医师，主要从事心血管疾病中西医结合诊疗的研究。 zhengjinghui@hotmail.com
  • 基金资助:

    课题为国家自然科学基金资助项目(30772696)，课题名称“养心通脉有效部位方对大鼠心肌缺血骨髓间充质干细胞移植的影响”；湖南研究生创新基金资助项目(CX2009B168)，课题名称“心血瘀阻证微环境变化及对骨髓间充质干细胞移植的影响”。

Effects of different serum microenvironments on culture of rat bone marrow mesenchymal stem cells in vitro  

Zheng Jing-hui, Li Yong-hua, Wang Li-ping, Jian Wei-xiong, Huang Xian-ping, Yuan Zhao-kai   

  1. Institute of Traditional Chinese Medicine Diagnosis, Hunan University of Traditional Chinese Medicine, Changsha   410007, Hunan Province, China
  • Online:2010-04-02 Published:2010-04-02
  • Contact: Yuan Zhao-kai, Professor, Institute of Traditional Chinese Medicine Diagnosis, Hunan University of Traditional Chinese Medicine, Changsha 410007, Hunan Province, China yuanzk1520@yahoo.com.cn
  • About author:Zheng Jing-hui☆, Studying for doctorate, Traditional Chinese Medicine Physician, Institute of Traditional Chinese Medicine Diagnosis, Hunan University of Traditional Chinese Medicine, Changsha 410007, Hunan Province, China zhengjinghui@hotmail.com
  • Supported by:

    the National Natural Science Foundation of China, No. 30772696*; the Innovation Foundation of Postgraduate of Hunan Province, No. CX2009B168*

PDF

484

被引次数

23

摘要：

背景：骨髓间充质干细胞在培养过程中，常在基础培养基中添加一定的血清，最常见的是小牛血清和胎牛血清，但这存在着一定的生物安全性隐患。
目的：观察不同血清微环境对大鼠骨髓间充质干细胞体外培养的影响。
方法：自成年大鼠骨髓中分离骨髓间充质干细胞，采用全骨髓贴壁法培养。分别以下列血清微环境培养：自身血清组：分离细胞后原代用含大鼠自身血清的培养基培养，传代后换用含胎牛血清的培养基培养；同种异体血清组：分离细胞后原代用含同种异体血清的培养基培养，以后用胎牛血清培养基培养；胎牛血清组：分离后用含胎牛血清培养基培养，以后一直用含胎牛血清培养基培养；DMEM组：分离细胞后原代用不含血清的DMEM培养基培养，传代后换用含胎牛血清的培养基培养。在倒置相差显微镜下观察细胞的形态；细胞的贴比率；生长曲线；流式细胞仪观测细胞表面CD11b、CD45和CD90表达。
结果与结论：大鼠自身血清微环境及同种异体血清微环境细胞形态均一性、在融合度均高于其他组；首次传代天数低于其他组。24，48，72 h贴比率自身血清组、同种异体血清组和胎牛血清组均高于DMEM组(P < 0.01)。在细胞生长曲线中大鼠自身血清组倍增速率最快，其次为同种异体血清组，再者为胎牛血清组，而DMEM组细胞未见明显倍增现象。流式细胞仪检测含血清培养基培养条件下各组第3代细胞CD11b和CD45阳性细胞率均达到98%以上，CD90阳性率小于2%，可以得到纯度较高的骨髓间充质干细胞。但是在无血清微环境条件下，CD11b的阳性率为95.83%、CD90阳性率达2.07%，而CD45阳性率仅为64.79%，可见无血清环境下培养的骨髓间充质干细胞纯度明显低于含血清微环境。提示大鼠自身血清微环境下更有利于骨髓间充质干细胞的贴壁、生长及纯化。

关键词: 微环境, 细胞培养, 骨髓间充质干细胞, 方法, 血清, 大鼠

Abstract:

BACKGROUND: During culture of bone marrow mesenchymal stem cells (BMSCs), a certain serum is commonly added in the basic medium, such as calf serum and fetal bovine serum, but there are potential biological safety risks.
OBJECTIVE: To study the effects of different serum microenvironments on in vitro culture of rat BMSCs.
METHODS: BMSCs were harvested from adult rat bone marrow, and cultured in vitro by whole bone marrow adherence method. The cells were cultured under the following serum microenvironment. The primary cells of autoserum group were cultured with autoserum, changing the medium with fetal bovine serum after passage. The primary cells of homogeneity foreign serum group were cultured with homogeneity foreign serum, changing the medium with fetal bovine serum after passage. The primary cells of fetal bovine serum group were cultured with fetal bovine serum, and cultured with fetal bovine serum after passage. The primary cells of Dulbecco's modified Eagle's medium (DMEM) group were cultured with serum-free DMEM, changing the medium with fetal bovine serum after passage. The morphologic changes in BMSCs were detected under an inverted phase contrast microscope. Attachment rate and growth curve were measured. Surface marker CD11b, CD45 and CD90 expression was analyzed by flow cytometry.
RESULTS AND CONCLUSION: In autoserum and homogeneity foreign serum groups, the homogenicity and degrees of fusion of cell morphology were improved in comparison with other two groups and the day of first passage was less than other groups. The attachment rate was greater in the autoserum, homogeneity foreign serum and fetal bovine serum groups than the DMEM group at 24, 48, 72 hours (P < 0.01). Doubling rate was fastest in the growth curve of autoserum group, followed by homogeneity foreign serum group and fetal bovine serum group. However, no doubling phenomenon was found in the DMEM group. Flow cytometry results demonstrated that the rates of CD11b-positive and CD45-positive cells at passage 3 were above 98% under medium containing serum, and CD90-positive rate was less than 2%. We could obtain BMSCs of higher purity. However, CD11b-positive rate was 95.83%, CD90-positive rate was 2.07%, but CD45 positive rate was only 64.79% under serum-free microenvironment. BMSC purity was significantly lower under serum-free microenvironment than under serum microenvironment. Results indicated that the microenvironment of rat autoserum can improve the attachment rate, growth and purification of BMSCs.

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