BMP9联合NGF对C3H10T1/2间充质干细胞骨向分化能力影响的实验研究

目的:探讨骨形态发生蛋白9(BMP9)与神经生长因子(NGF)联合处理对间充质干细胞(MSCs)骨向分化的影响.方法:以重组腺病毒法将BMP9导入C3H10T1/2细胞,构建MSCs骨向分化实验模型,设定分组为绿色荧光(GFP)对照组、NGF单独处理组、BMP9单独处理组以及BMP9+ NGF联合处理组,分别在处理3h、12 h、24 h、48 h、3d、7d后检测早期成骨分化标志物Ⅰ型胶原、Runt相关转录因子2(RUNX2)和碱性磷酸酶(ALP)在细胞中的表达.检测方法采用ALP染色、实时定量RT-PCR检测mRNA转录水平以及Western blot检测蛋白表达.结果:BMP9+ NGF组较各单独处理组ALP活性显著增加,Ⅰ型胶原、RUNX2的mRNA水平在BMP9+ NGF组的表达明显高于其他各单独处理组,且各组在3h即出现显著差异;RUNX2蛋白在各实验组均表达,BMP9+ NGF组表达最高.结论:BMP9联合NGF在成骨诱导间充质干细胞分化早期,协同促进其骨向分化.

The effects of BMP9 combined with NGF on the osteogenic differentiation of C3H10T1/2 mesenchymal stem cells

Objective:To investigate the effects of BMP9 combined with NGF on the osteogenic differentiation of mesenchymal stem cells(MSCs).Methods:Recombinant BMP9 adenovirus was transfected into C3H10T1/2 cells.The cells were treated by GFP,NGF,BMP9 and BMP9 + NGF respectively.The expression level of COL1,RUNX2 was detected by RT-PCR and Western blot,ALP activity was examined by ALP kit 3,12,24,48 hours,3 and 7 days after treatment,respectively.Results:The ALP activity of BMP9 + NGF group was the highest among the 4 groups.The difference in the groups firstly appeared at 3 h after treatment.The highest expression level of RUNX2 and COL1 was detected in BMP9 + NGF group.Conclusion:NGF and BMP9 may synergisticly promote osteogenic differentiation at the early stage of osteogenic induction of C3H10T1/2 cells.