天津医药

天津医药 ›› 2015, Vol. 43 ›› Issue (2): 142-146.doi: 10.11958/j.issn.0253-9896.2015.02.008

• 细胞与分子生物学 • 上一篇    下一篇

人脐带沃顿胶间充质干细胞体外分离条件的优化及传代效应的流式细胞学检测

洪敬欣1 ， 李茜1,2 ， 韩俊领1,2△   

  1. 1天津， 协和干细胞基因工程有限公司 （邮编300384）； 2中国医学科学院、 北京协和医学院， 血液学研究所， 血液病医院
  • 收稿日期:2014-07-31 修回日期:2014-09-22 出版日期:2015-02-15 发布日期:2015-02-27
  • 通讯作者: 韩俊领 E-mail:hjx78@126.com

Optimization the methodology of isolating human ubilical cord mesenchymal stromal cells from Wharton's jelly and examination of their passage effect on immune phenotype using flow cytometry

  • Received:2014-07-31 Revised:2014-09-22 Published:2015-02-15 Online:2015-02-27

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摘要： 摘要： 目的 观察不同胶原酶消化方法对人脐带沃顿胶间充质干细胞 （mesenchymal stem cell， MSC） 分离和培养结果的影响， 并鉴定其分化潜能； 探讨传代效应对其免疫表型的影响。方法 将制备好的脐带标本分别加入Ⅰ型、 Ⅱ型及Ⅳ型胶原酶， 持续消化 4～18 h， 筛网过滤， 离心收集细胞， 用 DMEM/F12 培养基重悬细胞， 调整细胞密度 4.8× 103 ～1×104 /cm2 ， 接种培养， 比较不同消化法分离人脐带沃顿胶 MSC 的效果。Von kossa 钙结节染色、 四环素荧光标记鉴定人脐带沃顿胶 MSC 向成骨方向分化的能力， RT-PCR 鉴定其向心肌细胞分化的能力。应用流式细胞仪检测连续传代后 MSC 的免疫表型变化。结果 Ⅰ型胶原酶消化法能够从人脐带沃顿胶获取了数量较多、 活力较高的 MSC， 而且细胞出现伸展的时间及原代培养时间均短于Ⅱ型胶原酶及Ⅳ型胶原酶消化法。表面标记分析显示： 随着传代次数的增加， 阳性标记 CD29、 CD44、 CD73、 CD90、 CD105 的表达百分率没有变化， 而阴性标记 CD31、 CD34 和 HLA-DR 的表达率增加明显。体外诱导实验表明： 来源于人脐带沃顿胶的 MSC 具有体外成骨和成心肌样细胞分化的能力。结论 Ⅰ型胶原酶消化法简单易行， 对细胞损伤小， 能稳定、 高效地从人脐带沃顿胶中分离出 MSC。

关键词: 脐带, 间质干细胞, 胶原酶类, 传代效应, 沃顿胶

Abstract: Abstract: Objective To observe the effects of different collagenase digestions on isolating human umbilical cord mesenchymal stromal cells (MSC) from Wharton’ s jelly, to exam their differentiation ability and to investigate their passage effect on the immune phenotype. Methods Human umbilical cord samples were digested by collagenaseⅠ or Ⅱor Ⅳ for 4-18 hours then were passed through sieves . Cells were collected by centrifugation then inoculated in DMEM/F12 medium at concentration within range of 4.8×103 -1×104 /cm2 to compare the effect of different digestions on MSC. Von kossa staining and tetracycline fluorescence was used to label the osteogenic differentiation capacity of MSC. Also RT-PCR was employed to identify the differentiate capacity of MSC into myocardial-like cells. The immunophenotype of MSCs were detected by flow cytometry after subculture. Results Using collagenaseⅠdigestion, the number of MSCs isolated from human umbilical cord in Wharton’ s jelly and their vitality were much higher while the period to show cell extension and primary culture time were shorter than those using collagenaseⅡor Ⅳ digestions. The analysis of surface marker revealed that the expression of positive markers include CD29, CD44, CD73, CD90 and CD105 did not change with passages while the negative markers such as CD31, CD34 and HLA-DR increased significantly with passages; Differential experiments induced in vitro show that human umbilical cord MSC in wharton’ s jelly had the ability to differentiate into osteoblasts and myocardial-like cells. Con⁃ clusion The human umbilical cord MSC in Wharton’ s jelly was successfully isolated by collagenaseⅠdigestion. This meth⁃ od was simple with a high success rate while cell loss and damage were minimum. This makes large-scale cultivation possi⁃ble. Negative markers increased with cell passages. This phenomenon revealed that MSC showed directional differentiation.

Key words: umbilical cord, mesenchymal stem cells, collagenases； passage effect, Wharton&rsquo, s jelly