Effects of fibroblast growth factor on proliferation and differentiation of serially passaged bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2017.25.001

Abstract

Abstract:

BACKGROUND: The source of bone marrow mesenchymal stem cells (BMSCs) is limited, and the cellular morphology, proliferation and multi-directional differentiation capacities can vary during serial passages in BMSCs in vitro.
OBJECTIVE: To study the effects of fibroblast growth factor (FGF) on cellular morphology, proliferation and differentiation of serially passaged BMSCs.
METHODS: (1) BMSCs were isolated from Sprague-Dawley rats and cultured. These cells were passaged six times in vitro, and the cellular morphology was observed and photographed. (2) BMSCs at passage 6 were seeded into 96-well plates and randomly divided into control group and FGF treatment group. The proliferation of cells in both groups was detected with cell counting kit-8 kit at days 1, 2, 3, 4, 5, 6, 7 after culture. (3) BMSCs at passage 6 were seeded into 6-well plates and randomly divided into control group and FGF treatment group. After 7 days treatment with growth medium or growth medium containing FGF, the cellular morphology was observed and photographed. And then the cells of both groups were treated with osteogenic induction medium, adipogenic induction medium and chondrogenic induction medium for the next 7 days. The osteogenic, adipogenic and chondrogenic related genes (RUNX2, ALP, OCN; PPARγ2, AP2, ADIPOQ; SOX9, collagen II, aggrecan) were detected with real-time PCR. The protein expressions of RUNX2, PPARγ2, SOX9 were detected with western blot assay. (4) BMSCs at passage 6 were seeded into 6-well plates and randomly divided into control group and FGF treatment group. After 7 days treatment with growth medium or growth medium containing FGF, the cells were cultured with osteogenic induction medium, adipogenic induction medium and chondrogenic induction medium for the next 14 days. Then, alizarin red S staining, oil red O staining and alcian blue staining were performed.
RESULTS AND CONCLUSION: (1) After in vitro passage for six times, the cellular morphology changed obviously, and FGF treatment recovered the characteristics of primary cells. (2) Compared with the control group, the cell proliferation in the FGF treatment group was significantly increased (P < 0.05). (3) Compared with the control group, the expression of osteogenic, adipogenic and chondrogenic related genes (RUNX2, ALP, OCN; PPARγ2, AP2, ADIPOQ; SOX9, collagen II, aggrecan) was increased significantly in the FGF treatment group (P < 0.05). The protein expressions of RUNX2, PPARγ2, SOX9 were also higher in the FGF treatment group than the control group (P < 0.05). (4) Compared with the control group, the number of extracellular calcium nodules, the number of intracellular lipid droplets, and the expression of acid acidic mucopolysaccharide were significantly increased after FGF pretreatment. To conclude, FGF pretreatment can preserve the stemness of BMSCs serially passaged in vitro.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Bone Marrow, Mesenchymal Stem Cells, Fibroblast Growth Factors, Cell Proliferation, Cell Differentiation, Tissue Engineering

CLC Number:

R394.2

Song Ming-yu, Yang Yong, Wu Hua, Wang Rong. Effects of fibroblast growth factor on proliferation and differentiation of serially passaged bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(25): 3937-3942.

Figures/Tables 2

2.1 成纤维细胞生长因子处理后能恢复骨髓间充质干细胞的形态原代培养的骨髓间充干细胞贴壁生长，细胞形态多为长梭形，少数呈多角形，折光性强，细胞铺满培养瓶底后呈漩涡状排列。传代培养到第6代时，细胞呈多角形、不规则形，折光性较弱，细胞排列不规则。用含10 μg/L成纤维细胞生长因子培养基培养7 d后，细胞形态恢复长梭形，折光性增强，并呈漩涡状排列(图1)。 2.2 成纤维细胞生长因子促进骨髓间充质干细胞增殖与用正常培养基培养的第6代骨髓间充干细胞相比，用含10 μg/L成纤维细胞生长因子的培养基培养的第6代骨髓间充干细胞增殖速度明显加快，从作用第3天开始，成纤维细胞生长因子的促增殖效应开始出现统计学差异，并持续到作用的第7天(图2)。 2.3 成纤维细胞生长因子处理后增强骨髓间充质干细胞向成骨、成脂、成软骨分化的能力与对照组相比，成纤维细胞生长因子处理组细胞成骨相关基因(RUNX2，ALP，OCN)、成脂相关基因(PPARγ2，AP2，ADIPOQ)、成软骨相关基因(SOX9，collagen 2，aggrecan)表达明显升高，差异有显著性意义(图3)；成纤维细胞生长因子处理组RUNX2、 PPARγ2 、SOX9蛋白表达明显高于对照组(图4)。第6代骨髓间充干细胞用含10 μg/L成纤维细胞生长因子的培养基预处理7 d，经成骨诱导培养基培养14 d后进行茜素红染色，与对照组相比，成纤维细胞生长因子预处理组细胞产生胞外钙结节的数量明显增加；经过成脂诱导培养基培养14 d后进行油红O染色，成纤维细胞生长因子预处理组细胞内脂滴的数量明显多于对照组；经过成软骨诱导14 d后进行阿利新蓝染色，成纤维细胞生长因子预处理组细胞酸性黏多糖的表达明显高于对照组(图5)。基因、蛋白和表观层面均表明，经过成纤维细胞生长因子预处理7 d后，第6代骨髓间充干细胞向成骨、成脂、成软骨分化能力均明显增强。"

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