中国组织工程研究

中国组织工程研究 ›› 2017, Vol. 21 ›› Issue (13): 2023-2028.doi: 10.3969/j.issn.2095-4344.2017.13.010

• 脐带脐血干细胞 umbilical cord blood stem cells • 上一篇    下一篇

培养体系对维持人脐带间充质干细胞特性及其体外扩增效率的影响

孙亚如，张炳强，王福斌，许  评，王二朴，李翠翠   

  1. 青岛市立医院卫生部细胞移植重点实验室临床中心，山东省青岛市  266000
  • 修回日期:2016-12-16 出版日期:2017-05-08 发布日期:2017-06-09
  • 通讯作者: 孙亚如，青岛市立医院卫生部细胞移植重点实验室临床中心，山东省青岛市 266000
  • 作者简介:孙亚如，男，1983年生，山东省青岛市人，汉族，2007年英国谢菲尔德大学毕业，硕士，主要从事干细胞相关技术的研究。 并列第一作者：张炳强，男，1979年生，山东省青岛市人，汉族，硕士，主要从事干细胞研究。

Effect of cultivation systems on the maintenance of human umbilical cord mesenchymal stem cell characteristics and their proliferation rate in vitro

Sun Ya-ru, Zhang Bing-qiang, Wang Fu-bin, Xu Ping, Wang Er-pu, Li Cui-cui   

  1. Cell Transplantation Laboratory of Ministry of Health, Qingdao Municipal Hospital, Qingdao 266000, Shandong Province, China
  • Revised:2016-12-16 Online:2017-05-08 Published:2017-06-09
  • Contact: Sun Ya-ru, Cell Transplantation Laboratory of Ministry of Health, Qingdao Municipal Hospital, Qingdao 266000, Shandong Province, China
  • About author:Sun Ya-ru, Master, Ministry of Health Cell Transplantation Laboratory, Qingdao Municipal Hospital, Qingdao 266000, Shandong Province, China Zhang Bing-qiang, Master, Cell Transplantation Laboratory of Ministry of Health, Qingdao Municipal Hospital, Qingdao 266000, Shandong Province, China Sun Ya-ru and Zhang Bing-qiang contributed equally to this work.

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摘要：

文章快速阅读：

文题释义：
脐带间充质干细胞的特性：脐带来源间充质干细胞的原始丰度高于骨髓、骨膜、胎盘来源间充质干细胞，其数量依然需要经过体外数代扩增才能达到临床应用的需求。然而，脐带间充质干细胞并非永生化的干细胞，在多次传代后，会随扩增的数量增多而逐渐分化，最终丧失干细胞特性，进而失去治疗功能。因此选择能最大限度维持干细胞特性及高扩增效率的培养体系至关重要。
无血清培养体系：是一种无任何血清成分，通过像鸡尾酒式的添加营养成分和生长因子就可以维持间充质干细胞在体外生长繁殖的培养体系。它不仅能够避免异种血清可能对细胞带来的异源性污染，而且较胎牛血清和人血小板裂解液培养体系在细胞增殖效率方面有较大提升。尽管如此，实验数据显示单独应用无血清培养体系作为临床级脐带间充质干细胞的培养体系尚有其不足之处：细胞在无血清培养体系中随传代次数递增容易出现分化，难以维持干细胞的特性；随传代数次递增扩增效率显著下降。

 

摘要
背景：前期数据显示，应用人血小板裂解液培养体系对人脐带间充质干细胞进行体外培养时，较无血清培养体系能更好地维持干细胞特性，而无血清培养体系相较于人血小板裂解液培养体系能一定程度上提高间充质干细胞的体外增殖能力。
目的：筛选能最大限度维持人脐带间充质干细胞特性及扩增效率高的培养体系。
方法：将6份人脐带标本分别接种于6种培养体系中，进行脐带间充质干细胞原代培养，6种培养体系分别为MesenCult无血清完全培养基中添加2%人血小板裂解液(A组)、StemPro无血清完全培养基中添加2%人血小板裂解液(B组)、MesenCult无血清完全培养基(C组)、StemPro无血清完全培养基(D组)、低糖DMEM中添加体积分数10%胎牛血清(E组)、低糖DMEM中添加10%人血小板裂解液(F组)，14 d后进行消化传代培养，比较各培养体系对人脐带间充质干细胞形态、表面标记物、分化及增殖能力的影响。
结果与结论：①D组第3代细胞细长，大小不均；E、F组第3代细胞扁平，其余组第3代细胞为长梭形且大小较均一。A、B组第5代细胞形态及大小较第3代时无明显变化，C组第5代细胞趋于扁平且大小不均一，D组第5代细胞形态较第3代时细长，E组第5代细胞形态较第3代时扁平，F组第5代细胞形态较第3代时无明显变化；②各组细胞表面标志物检测结果无明显区别；③A、B组成脂、成骨诱导率较高，E、F组次之，C、D组最低；④A组各代次细胞扩增数量显著高于C组，B组各代次细胞扩增数量显著高于D组，E、F组扩增数量显著低于培养组A、B组；⑤结果表明，无血清和人血小板裂解液相结合的培养基能较好地维持间充质干细胞的特性及扩增效率。

 

 

关键词: 干细胞, 脐带脐血干细胞, 脐带, 间充质干细胞, 培养体系, 培养基, 筛选, 形态, 表型, 分化, 扩增

Abstract:

BACKGROUND: Preliminary data showed that the application of human platelet lysate to human umbilical cord mesenchymal stem cell culture can better maintain the characteristics of stem cells than the application of serum-free medium. However, the serum-free medium can better improve the proliferation of mesenchymal stem cells in vitro than the human platelet lysate.
OBJECTIVE: To screen out a better mesenchymal stem cell cultivation system that can greatly maintain the characteristics and proliferation rate of human umbilical cord mesenchymal stem cells.
METHODS: Six human umbilical cord specimens were inoculated in six culture systems, and the primary culture of umbilical cord mesenchymal stem cell was performed. These six culture systems were respectively MesenCult serum-free medium with 2% human platelet lysate (group A), StemPro serum-free medium with 2% human platelet lysate (group B), MesenCult serum-free medium (group C), StemPro serum-free medium (group D), low glucose-DMEM with 10% fetal bovine serum (group E), low glucose-DMEM with 10% human platelet lysate (group F). The cells were subcultured at 14 days after inoculation to compare the effects of different culture systems on the morphology, surface markers, differentiation and proliferation of human umbilical cord mesenchymal stem cells.
RESULTS AND CONCLUSION: (1) The morphology of passage 3 cells in group D was elongated and uneven in size. The morphology of passage 3 cells was flattened in groups E and F, but the cells in the other groups were spindle-shaped and uniform. There were no significant changes in morphology and size between passage 3 and 5 cells in A and B. In group C, the morphology of passage 5 cells was more flattened and uneven in size compared with passage 3 cells. In group D, the morphology of passage 5 cells was more elongated than that of passage 3 cell. In group E, the morphology of passage 5 cells was more flattened than that of passage 3 cells. There was no significant difference in morphology between passage 3 and 5 cells in group F. (2) The expression rate of cell surface markers had no significant difference at different passages in each group. (3) The adipoinduction and osteoinduction rates were relatively higher in groups A and B compared with groups E and F, and lowest in groups C and D. (4) The cell proliferation rate for each passages in group A was significantly higher than that in group C. The cell proliferation rate for each passage in group B was significantly higher than that in group D. The cell proliferation rate for each passage in groups E and F was significantly lower than that in groups A and B. To conclude, these results suggest that the combination of serum-free medium with human platelet lysate could better maintain the characteristics and the proliferation efficiency of mesenchymal stem cells.

 

 

Key words: Stem Cells, Umbilical Cord, Mesenchymal Stem Cells, Tissue Engineering

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