两种细胞因子联合应用对骨髓基质干细胞增殖和成骨活性的影响

Effects of fusion bone morphogenetic proteins and basic fibroblast growth factors on proliferation and osteogenic activity of rabbit bone mesenchymal stem cells （BMSCs） in vitro

目的 研究骨形态发生蛋白融合基因-4/7（BMP-4/7）和碱性成纤维细胞生长因子（bFGF）联合应用对体外培养兔骨髓基质干细胞（BMSCs）增殖和成骨活性的影响.方法 体外培养BMSCs,在第3代细胞培养液中加入不同浓度的BMP-4/7和bFGF,依据加入不同基因浓度组合的不同分为5个实验组（A组：80 ng/mL BMP-4/7,B组：80 ng/mL bFGF,C组：30 ng/mL BMP-4/7＋30ng/mL bFGF,D组：50 ng/mL BMP-4/7＋50 ng/mL bFGF,E组：80 ng/mL BMP-4/7＋80 ng/mL bFGF）和对照组（不加任何因子）,采用绘制生长曲线,甲基噻唑基四唑（MTT）比色法检测细胞增殖活力,碱性磷酸酶（ALP）和降钙素（OC）活性检测法比较各组间差异,观察不同浓度的BMP-4/7和bFGF联合应用对兔BMSCs增殖和成骨活性的影响. 结果 传代后第5天对照组个别单核细胞贴壁,呈长梭形 A组细胞增殖,呈旋涡状排列 B组细胞较为密集,部分融合成片 C组细胞呈集落式生长,生长旺盛 D组细胞生长密集,可见明显的钙结节 E组细胞密集,可见细胞性钙结节形成.各组OD值、ALP含量、OC含量随着作用时间的延长而增加,各组不同培养时间的OD值差异均有统计学意义（P〈0.01） 且C、D、E组均高于A、B组,差异均有统计学意义（P〈0.05）.C、D、E组内随着作用浓度的增加,细胞增殖及成骨活性增强,呈浓度依赖关系,差异均有统计学意义（P〈0.05）. 结论 合理的联合应用BMP-4/7和bFGF可促进BMSCs细胞增殖,促进成骨活性,两者对BMSCs有明显的协同增强效应.

Objective To study the combined effects of basic fibroblast growth factor （bFGF） and fusion bone morpbogenetic gene protein-4/7 （BMP-4/7） on proliferation and osteogenic activity of rabbit bone mesenchymal stem cells （BMSCs） in vitro. Methods BMSCs cells were cultured in vitro. Different concentrations of BMP-4/7 and bFGF were added into the culture of the third passage. Five experimental groups were established according to the concentration of BMP-4/7 and bFGF. Group A： 80 ng/mL BMP-4/7 group B： 80 ng/mL bFGF group C： 30 ng/mL BMP-4/7 ＋30 ng/mL bFGF group D： 50 ng/mL BMP-4/7 ＋50 ng/mL bFGF group E： 80 ng/mL BMP-4/7 ＋80 ng/mL bFGF） . No growth factor was added into the culture fluid in the control. The growth curve drawing, methyl thiazolyl tetrazolium （MTT）, alkaline pbosphatase （ALP） and osteocalcin （OC） dynamics were used to compare the differences between the groups and observe the effects of BMP-4/7 plus bFGF on proliferation and osteogenic differentiation of BMSCs. Results Compared with the control group, single BMP-4/7 had promotive effects on the pro-liferation and osteogenic differentiation of BMSCs, while single bFGF contributed to the proliferation of BMSCs significandy hut inhibited the osteogenic differentiation of BMSCs. The combination of BMP-4/7 and bFGF promoted not only the BMSCs proliferation significantly but also the osteogenic differentiation of BMSCs,showing positive correlation with action time and concentration. Conclusion A rational combined use of BMP-4/7 and bFGF can promote the proliferation and osteogenic differentiation of BMSCs and the two factors have significant synergistic effect on BMSCs.