成纤维生长因子-2联合骨形态发生蛋白-2体外诱导大鼠骨髓间充质干细胞向心肌样细胞的分化

Bone morphogenetic protein-2 combined with fibroblast growth factor-2 induce bone marrow mesenchymal stem cells in vitro to differentiate into cardiomyocytes in rats

目的:观察纤维生长因子-2(FGF-2)与骨形态发生蛋白-2(BMP-2)单一及联合诱导大鼠骨髓间充质干细胞(BMMSCs)向心肌样细胞分化的效果。方法:从SD大鼠骨髓中分离提取BMMSCs,细胞传至第2代时加入诱导物质,分组(FGF-2诱导组、BMP-2诱导组、联合诱导组及空白对照组)培养。应用免疫细胞化学法对培养至第28天时4组细胞中cTnT、desmin、α-actin、p38mapk蛋白表达情况进行检测;应用RT-PCR法对4组细胞中GATA-4、Nkx2.5及α-MHC mRNA的表达量进行测定。结果:常规养至第28天时,联合诱导组细胞中cTnT、desmin、α-actin和p38mapk蛋白表达的阳性率依次为(30.14±8.66)%、(45.30±6.96)%、(60.95±7.73)%和(43.50±7.32)%,且FGF-2诱导组、BMP-2诱导组各指标的阳性率均低于联合诱导组(P<0.05)。FGF-2诱导组、BMP-2诱导组细胞中Nkx2.5及GATA-4 mRNA的表达量在第7、14、28天均呈现"高-低-高"的变化趋势;而联合诱导组中以上两种基因的表达量呈现"低-高-高"的表达趋势;且第28天时联合诱导组中以上两种基因的表达量明显高于单一诱导组(P<0.05);各组细胞均未见α-MHC mRNA表达。结论:BMMSCs可在FGF-2与BMP-2单一及联合诱导下发育为心肌样细胞,且两者联合诱导效果更佳。

Aim： To investigate the effect of fibroblast growth factor-2（ FGF-2） or bone morphogenetic protein-2（ BMP-2） or their combination on the differentiation of rat bone marrow mesenchymal stem cells（ BMMSCs） into cardiomyocyte-like cells. Methods： BMMSCs were isolated from SD rats and cultured with whole bone marrow adherence method. The second generation BMMSCs were co-incubated for 3 d with FGF-2 or BMP-2 or their combination. The control group was cultured without any inducer. At cells were cultured for 28 d,the positive rate of cTnT,desmin,α-actin and p38 mapk were determined by immunocytochemistry. Meanwhile,mRNA expressions of α-MHC,Nkx2. 5 and GATA-4 in BMMSCs were determined by RT-PCR on the 7 th,14 th and 28 th day after induction. Results： When BMMSCs being incubated by FGF-2 or BMP-2 or their combination and cultured for 28 d,the positive rates of cTnT,desmin,α-actin and p38 mapk in the combined induction group were（ 30. 14 ± 8. 66） %,（ 45. 30 ± 6. 96） %,（ 60. 95 ± 7. 73） %,（ 43. 50 ± 7. 32） %,respectively,moreover the rate higher than those in FGF-2 or BMP-2 induction group（ P〈0. 05）. RT-PCR results showed that cells in 4 groups did not express α-MHC. At cells were cultured for 28 d,the expression of GATA-4 and Nkx2. 5 compared with the control group were all obviously higher after BMMSCs were induced by BMP-2 or FGF-2 or their combination,in addition the combined induction of both FGF-2 and BMP-2 has a synergistic effect on the expression of each gene（ P〈0. 05）. Conclusion： BMMSCs could differentiate into cardiomyocyte-like cells under the induction of FGF-2,BMP-2 and their combination in vitro,and FGF-2 combined with BMP-2 may attain better results than the single inducer.