纤维蛋白凝胶复合碱性成纤维细胞生长因子与大鼠胎肢细胞碱性磷酸酶的活性

doi:10.3969/j.issn.1673-8225.2010.47.045

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (47): 8927-8930.doi: 10.3969/j.issn.1673-8225.2010.47.045

• 组织工程骨及软骨材料 tissue-engineered bone and cartilage materials • 上一篇

纤维蛋白凝胶复合碱性成纤维细胞生长因子与大鼠胎肢细胞碱性磷酸酶的活性

王英慧，陈莉，买霞，扎拉嘎胡，陈小义

武装警察部队医学院细胞生物学与医学遗传学教研室，天津市 300162

出版日期:2010-11-19 发布日期:2010-11-19
通讯作者: 陈莉，硕士，教授，硕士生导师，武装警察部队医学院细胞生物学与医学遗传学教研室，天津市 300162
作者简介:王英慧★，女，1975年生，河北省丰润县人，汉族，河北医科大学在读硕士，实验师，主要从事干细胞定向分化研究。
基金资助:
武装警察部队资助重点项目(WKH2007-1)。

Effects of fibrin gel combined with basic fibroblast growth factor on alkaline phosphatase activity in fetal rat limb cells

Wang Ying-hui, Chen Li, Mai Xia, Zha La Ga Hu, Chen Xiao-yi

Department of Cell Biology and Medical Genetics, Medical College of Chinese People's Armed Police Forces, Tianjin 300162, China

Online:2010-11-19 Published:2010-11-19
Contact: Chen Li, Master, Professor, Master’s supervisor, Department of Cell Biology and Medical Genetics, Medical College of Chinese People's Armed Police Forces, Tianjin 300162, China chenli054@yahoo.com.cn
About author:Wang Ying-hui★, Studying for master’s degree, Experimentalist, Department of Cell Biology and Medical Genetics, Medical College of Chinese People's Armed Police Forces, Tianjin 300162, China diandema@sohu.com
Supported by:
Emphasis Program of Chinese People’s Armed Police Forces, No. WKH2007-1*

摘要/Abstract

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。碱性成纤维细胞生长因子是一种对中胚层和神经外胚层细胞促分裂作用的多肽生长因子。两者结合有利于干细胞在高度交联的凝胶支架内迁移，并且促进其增殖与分化。
目的：观察纤维蛋白凝胶复合碱性成纤维细胞生长因子对大鼠胎肢细胞碱性磷酸酶活性的影响。
方法：实验分为4组：纤维蛋白凝胶组，纤维蛋白凝胶中加入正常培养基。纤维蛋白凝胶复合碱性成纤维细胞生长因子组，在纤维蛋白凝胶中加入含10 μg/L碱性成纤维细胞生长因子培养基。碱性成纤维细胞生长因子组，加入10 μg/L 碱性成纤维细胞生长因子培养基。对照组，采用无凝胶正常培养基培养。无菌条件下分离培养大鼠胎肢细胞，取第3代细胞接种于以上培养基中。分别在第3, 5, 7, 14, 21, 28天检测碱性磷酸酶活性、mRNA表达及细胞形态观察。
结果与结论：在有纤维蛋白凝胶组内培养至7 d细胞具有较长突起且连接成网，而在无纤维蛋白凝胶组内的细胞呈纺锤形或立方形；纤维蛋白凝胶复合碱性成纤维细胞生长因子培养7，14，21 d碱性磷酸酶活性高于单纯纤维蛋白凝胶组和单纯碱性成纤维细胞生长因子培养组(P < 0.05)。各时间点纤维蛋白凝胶复合碱性成纤维细胞生长因子组碱性磷酸酶mRNA表达均较对照组增强(P < 0.01)。提示纤维蛋白凝胶复合碱性成纤维细胞生长因子有利于细胞形态发生，并明显增强碱性磷酸酶活性。

关键词: 纤维蛋白凝胶, 碱性成纤维细胞生长因子, 碱性磷酸酶, 细胞培养, 生物材料

Abstract:

BACKGROUND: Fibrin is a natural biodegradable polymer material with good histocompatibility. Basic fibroblast growth factor (bFGF) is an important polypeptide growth factor with mitogenic effect. Fibrin gel (FG) combined with bFGF is conducive to cell migration and promotes proliferation and differentiation.
OBJECTIVE: To investigate the effects of fibrin gel combined with bFGF on alkaline phosphatase activity (ALP) in fetal rat limb cells.
METHODS: Four groups were included: FG group: fetal rat limb cells were seeded in FG (three-dimensional, 3D) with normal medium; FG + bFGF group: cells were seeded in FG containing 10 μg/L bFGF for 24-hour 3D culture; bFGF group: cells were seeded in 10 μg/L bFGF medium; normal control group: cells were seeded in normal culture medium. Under aseptic condition, rat fetal limb cells were isolated. Passage 3 cells were used for each group above mentioned. ALP activity, mRNA expression and cell morphology were analyzed at 3, 5, 7, 14, 21, and 28 days after culture.
RESULTS AND CONCLUSION: The cells in the FG had more processes and formed an interconnected network after 7 days of incubation, while the cells in no gels remained cuboidal or “spindle-shaped”. At 7, 14, and 21 days, ALP activity was greater in the FG+ bFGF group than in the other groups (P < 0.05). At each time point, mRNA expression of ALP was greater in the FG+ bFGF group than in the control group (P < 0.01). These findings indicate that FG combined with bFGF is conducive to cell morphogenesis and markedly enhances ALP activity.

中图分类号:

R394.2

王英慧，陈莉，买霞，扎拉嘎胡，陈小义. 纤维蛋白凝胶复合碱性成纤维细胞生长因子与大鼠胎肢细胞碱性磷酸酶的活性[J]. 中国组织工程研究, 2010, 14(47): 8927-8930.

Wang Ying-hui, Chen Li, Mai Xia, Zha La Ga Hu, Chen Xiao-yi. Effects of fibrin gel combined with basic fibroblast growth factor on alkaline phosphatase activity in fetal rat limb cells[J]. Chinese Journal of Tissue Engineering Research, 2010, 14(47): 8927-8930.

参考文献

引言

材料方法

讨论

FG is a natural biodegradable polymer material with good histocompatibility. It is also a carrier to promote the release of intracellular and exogenous growth factor. Therefore, it is a good structure engineering bracket material[6-9]. bFGF is an important polypeptide growth factor with mitogenic effect on the cells from mesoderm and neuroectoderm tissue, and affects cell adhesion, proliferation and differentiation[10-13]. It enhances the ability of osteocytes to synthesize collagen and other proteins[14], promotes sclerotin growth, and is one of crucial wound healing factors in vivo[15-16]. Results from this study indicate that FG combined with bFGF promotes cell migration in cross-linked FG and enhances ALP activity of cells.
ALP, a functional enzyme in osteoblasts, plays a crucial role in calcium deposition. ALP can hydrolyze organophosphates, which increases the concentration of local phosphate groups, and also can block calcification inhibitors to activate calcification. Therefore, enhancement in ALP activity is an important indicator of differentiation of stem cells into osteoblasts.
Cellular ALP activity results demonstrated that the absorbance value in the normal control group was not increased before 21 days and began to increase after 21 days. ALP activity in the FG group began to increase after 7 days of culture, with the absorbance value gradually increased with culture time. At 21 days, the absorbance value in the FG group was 21% higher than the normal control group, which is consistent with previous reports[4, 17-19]. These findings suggest that FG is conducive to cell differentiation and can maintain ALP activity at a high level.
After addition of bFGF into the FG, the absorbance value began to increase at 3 days and was gradually increased with the culture time, peaked at 21 days. The peak level of absorbance value in the FG + bFGF group was 82% higher than the normal control group, 51% higher than the FG group, and 26% higher than the bFGF group. RT-PCR results regarding ALP mRNA expression further confirmed the detection results of ALP activity, suggesting that three-dimensional culture of FG and bFGF is more conducive to cell differentiation. Evidence exists that the matrix regulatory role of adult mesenchymal stem cells in the in vitro differentiation of osteoblasts and the ability of in vivo bone formation are very important[20-21] and suggests that addition of appropriate factors in the matrix facilitates the maintenance of mesenchymal stem cell differentiation potential.
Taken together, the present study demonstrated that the ALP activity increased earlier and maintained a high level over a long period after combining FG and bFGF, because bFGF could promote stem cell proliferation and differentiation within the FG matrix, a biological material. RT-PCR experiments further confirmed that bFGF up-regulated the ALP mRNA expression of fetal rat limbs cells. These findings provide theoretical foundation for investigating tissue engineering application in the wound healing.

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纤维蛋白凝胶复合碱性成纤维细胞生长因子与大鼠胎肢细胞碱性磷酸酶的活性

Effects of fibrin gel combined with basic fibroblast growth factor on alkaline phosphatase activity in fetal rat limb cells

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