中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (23): 4220-4224.doi: 10.3969/j.issn.1673-8225.2011.23.010

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

人脐带来源间充质干细胞分离培养方法的优化

齐  凯，董丽媛，陈显久，赵  婕，薛国芳，陈  彦，牛  勃   

  1. 山西医科大学生物化学与分子生物学教研室，山西省太原市030020
  • 收稿日期:2011-01-04 修回日期:2011-03-16 出版日期:2011-06-04 发布日期:2011-06-04
  • 通讯作者: 牛勃，博士，教授，博士生导师，山西医科大学生物化学与分子生物学教研室，山西省太原市 030020 niub2004@126.com
  • 作者简介:齐凯★，男，1986年生，山西省临汾市人，山西医科大学基础医学院在读硕士，主要从事蛋白质结构与功能研究。 angelqikai@163.com
  • 基金资助:

    国家自然科学基金(30600226，30472251)。

Optimized study on isolation and cultivation of human umbilical cord mesenchymal stem cells

Qi Kai, Dong Li-yuan, Chen Xian-jiu, Zhao Jie, Xue Guo-fang, Chen Yan, Niu Bo   

  1. Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan 030020, Shanxi Province, China
  • Received:2011-01-04 Revised:2011-03-16 Online:2011-06-04 Published:2011-06-04
  • Contact: Niu Bo, Doctor, Professor, Doctoral supervisor, Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan 030020, Shanxi Province, China niub2004@126.com
  • About author:Qi Kai★, Studying for master’s degree, Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan 030020, Shanxi Province, China angelqikai@163.com
  • Supported by:

    the National Natural Science Foundation of China, No. 30600226*, 30472251*

PDF

457

被引次数

16

摘要：

背景：关于人脐带间充质干细胞的分离培养方法不一，如何提高人脐带来源间充质干细胞获得效率的问题尚未解决。
目的：优选人脐带来源间充质干细胞制备的消化酶组分。
方法：无菌条件下取正常足月剖腹产的脐带近胎儿段，按不同混合酶浓度比值划分为3组，混合酶Ⅰ组成分为0.4%Ⅱ型胶原酶、0.1%胰酶、0.02%EDTA和0.1%透明质酸酶；混合酶Ⅱ组成分为0.1%Ⅱ型胶原酶、0.4%胰酶、0.02%EDTA和0.1%透明质酸酶；混合酶Ⅲ组成分为0.3%Ⅱ型胶原酶、0.1%胰酶、0.02%EDTA、0.1%透明质酸酶和0.1%DNA酶。每组再按消化时间分为1，2，3 h 3个亚组。最后重悬细胞终体积均定位4 mL并计数，采用含血清的DMEM培养液体外培养细胞。
结果与结论：采用3种不同混合酶浓度进行消化，发现相同消化时间内，混合酶Ⅲ组消化细胞最多(P < 0.05)。在作用3 h时，混合酶Ⅲ组获取的细胞中活细胞比值显著低于混合酶Ⅰ组和混合酶Ⅱ组(P < 0.01)。提示0.3%Ⅱ型胶原酶、0.1%胰酶、0.02%EDTA、0.1%透明质酸酶和0.1%DNA酶混合液消化脐带组织块2 h为获取脐带间充质干细胞的最佳条件。

关键词: 脐带, 间充质干细胞, Ⅱ型胶原酶, 透明质酸酶, DNA酶Ⅰ

Abstract:

BACKGROUND: There are different methods to isolate and culture human umbilical cord mesenchymal stem cells (hUC-MSCs), so how to fast and efficiently harvest hUC-MSCs have become a research hotspot.
OBJECTIVE: To optimize digestive enzymes components for the preparation of hUC-MSCs, in order to lay the foundation of preparation for hUC-MSCs.
METHODS: Human umbilical cords were collected from full term deliveries under aseptic conditions. According to different mixed enzyme concentration ratio, the samples were divided into three groups: mixed enzyme Ⅰ group, mixed enzyme Ⅱ group, mixed enzyme Ⅲ group. Then according to digest time each group into three subgroups: 1 hour, 2 hours, and 3 hours. Finally, suspended cell volume was decided as 4 mL to count cells. Dulbecco’s Modified Eagle Medium with fetal bovine serum was used for cell culture.
RESULTS AND CONCLUSION: Based on the three digestion enzyme concentration, at the duration of 1 hour, 2 hours and 3 hours, mixed enzyme Ⅲ group had the highest total cell number and the total cell rate was statistically different from the other groups (P < 0.05). At the duration of 3 hours, live cell rate was the lowest in the mixed enzyme Ⅲ group, and there was statistically significant differences among these groups (P < 0.01). The optimal isolation for hUC-MSCs is 0.3% collagenase Ⅱ, 0.1% trypsin, 0.02% EDTA, 0.1% hyaluronidase and 0.1% DNA enzymeⅠ digestion for 2 hours.

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