中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (12): 2091-2094.doi: 10.3969/j.issn.1673-8225.2010.12.001

• 组织工程骨及软骨材料 tissue-engineered bone and cartilage materials •    下一篇

纤维蛋白凝胶对大鼠胚胎间充质干细胞增殖与成骨分化的影响：不同质量浓度比较

买  霞，陈  莉，陈小义，扎拉嘎胡   

  1. 武装警察部队医学院细胞生物学教研室，天津市 300162
  • 出版日期:2010-03-19 发布日期:2010-03-19
  • 通讯作者: 陈 莉，教授，硕士生导师，武装警察部队医学院细胞生物学教研室，天津市 300162 chenli054@yahoo.com.cn
  • 作者简介:买 霞，女，1972年生，山西省运城市人，回族，1998年天津医科大学毕业，讲师，主要从事干细胞定向分化方面的研究。 maixia1212@163.com
  • 基金资助:
    武警部队资助重点项目(WKH2009Z04)，课题名称“可注射性组织工程软骨促进关节软骨缺损的愈合”。

Effects of fibrin gel on proliferation and osteogenic differentiation of rat embryonic mesenchymal stem cells: A comparison among various concentrations

Mai Xia, Chen Li, Chen Xiao-yi, Zha La-ga-hu   

  1.  Department of Cell Biology, Medical College of Chinese People’s Armed Police Forces, Tianjin   300162, China
  • Online:2010-03-19 Published:2010-03-19
  • Contact: Chen Li, Professor, Master’s supervisor, Department of Cell Biology, Medical College of Chinese People’s Armed Police Forces, Tianjin 300162, China chenli054@yahoo.com.cn
  • About author:Mai Xia★, Lecturer, Department of Cell Biology, Medical College of Chinese People’s Armed Police Forces, Tianjin 300162, China maixia1212@163.com
  • Supported by:

    the Major Program of Chinese People＇s Armed Police Forces, No. WKH2009Z04*

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摘要：

背景：纤维蛋白是一种天然的可生物降解、组织相容性好的高分子材料，其中血纤维蛋白稳定因子ⅩⅢ已经证明有利于未分化间充质干细胞在高度交联的凝胶支架内迁移，并促进其增殖与分化。
目的：探讨大鼠胚胎间充质干细胞在不同质量浓度纤维蛋白凝胶内的形态学变化，以及生长增殖和成骨分化情况。
方法：无菌条件下取胎鼠四肢，组织块消化法体外分离培养胚胎间充质干细胞。取传至第3代细胞，分别接种于20，10，5 g/L纤维蛋白凝胶内，并设立对照组，接种于无凝胶的正常培养基中。用激光扫描共聚焦显微镜观察细胞在凝胶内的形态学变化，荧光分光光度计测定细胞增殖状态，酶标仪和Von Kossa染色分析细胞碱性磷酸酶活性和钙盐沉积情况。
结果与结论：培养7 d，在纤维蛋白凝胶内的大鼠胚胎间充质干细胞具有较长突起，且连接成网，而对照组细胞呈纺锤形或立方形。与对照组比较，凝胶组细胞增殖活跃，至培养第14天达峰值，以5 g/L凝胶细胞组荧光最强；凝胶组细胞碱性磷酸酶活性从14 d开始增强，21 d达峰值。培养14 d后20 g/L凝胶组在凝胶区出现小矿化结节，随后逐渐融合，而对照组无矿化结节出现。证实纤维蛋白凝胶支架能够支持大鼠胚胎间充质干细胞的存活与生长，且可以促进细胞的增殖和分化。   5 g/L低浓度凝胶有利于细胞形态的发生，20 g/L高浓度凝胶有利于细胞的成骨分化。

关键词: 纤维蛋白凝胶, 细胞增殖, 细胞分化, 碱性磷酸酶, 细胞培养, 胚胎, 间充质干细胞

Abstract:

BACKGROUND: Fibrin is a kind of high polymer materials with biodegradation and good histocompatibility. Fibrin stabilizing factor ⅩⅢ has been verified that it can contribute to the migration of undifferentiated mesenchymal stem cells (MSCs) in gel scaffold with high crosslinking, and promote its proliferation and differentiation.
OBJECTIVE: To analyze morphological changes of rat embryonic MSCs in fibrin gel of different mass concentrations, and to observe the growth, proliferation and osteogenic differentiation.
METHODS: To isolate and culture fetal limbs mesenehymal stem cells (flMSCs) under aseptic condition using tissue digesting method. The third passage of flMSCs were incubated in three different concentrations (20, 10 and 5 g/L) of fibrin gels. A control group was set and incubated in normal medium without gel. The cell morphology in fibrin gels was observed by confocal laser scanning microscope. Cell proliferation was assessed by fluorospectrophotometer. A microplate reader and Von Kossa staining were used to analyze alkaline phosphatase (ALP) activity and calcium salts mineralization.
RESULTS AND CONCLUSION: FlMSCs had long processes which connected each other and formed network in fibrin gels, but fusiform or cube cells were observed in the control group at 7 days. Compared with the control group, cell proliferation was great in the fibrin gel group, and peaked at day 14, especially in the 5 g/L fibrin gel group. ALP activity was increased at day 14 in the fibrin gel group, and reached a peak at day 21. Following 14 days of incubation, mineralization was observed in 20 g/L fibrin gel group, and then gradually fused. However, no mineralization was detected in the control group. Results verified that fibrin gel scaffold can support the survival and growth of rat embryonic MSCs, and promote cell proliferation and differentiation. 5 g/L fibrin gel contributes to cell morphological changes, and 20 g/L fibrin gel contributes to osteogenic differentiation.

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