论文部分内容阅读
为了探讨骨形态发生蛋白4(bone morphogenetic protein 4,BMP4)在诱导骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)向生殖细胞分化中的作用,将全骨髓培养法获取小鼠BMSCs,在含10%胎牛血清(fetal bovine serum,FBS)的DMEM培养液中培养传代;取生长状态良好的第三代(P3)BMSCs,分别添加不同浓度(5、10、20、40和80ng·mL~(-1))的BMP4作为实验组,未添加BMP4为对照组。4d后,MTT法和台盼蓝染色检测细胞的增殖率和存活率,实时定量PCR(RT-qPCR)检测生殖细胞阶段特异性基因(Oct4、Dazl、Fragilis、Mvh、Nobox、Stella、Stra8和Gdf9)的表达。结果发现,BMP4浓度为5~20 ng·mL~(-1)时细胞存活率和增殖率最高;BMP4浓度为20 ng·mL~(-1)组Mvh、Fragilis、Oct-4、Dazl及Stella表达水平最高,均显著高于对照组(P<0.05);Nobox表达水平则在BMP4浓度40ng·mL~(-1)组最高,且各浓度组均显著高于对照组(P<0.05);各浓度BMP4组Stra8和Gdf9的表达水平均与对照组间无显著性差异(P>0.05)。此结果提示,BMP4浓度在20 ng·mL~(-1)时,BMSCs的存活率、增殖率及原始生殖细胞特异性基因的表达最高;BMP4在诱导间充质干细胞向生殖细胞分化过程中起重要作用。
To investigate the role of bone morphogenetic protein 4 (BMP4) in inducing the differentiation of bone marrow mesenchymal stem cells (BMSCs) into germ cells, mouse BMSCs were obtained by whole bone marrow culture. The third generation (P3) BMSCs with good growth status were cultured in DMEM containing 10% fetal bovine serum (FBS), and the cells were cultured in different concentrations (5, 10, 20, 40 and 80 ng · mL ~ (-1)) BMP4 as experimental group, without adding BMP4 as control group. After 4 days, the cell proliferation rate and survival rate were detected by MTT assay and trypan blue staining, and the germ cell stage specific genes (Oct4, Dazl, Fragilis, Mvh, Nobox, Stella, Stra8 and Gdf9 were detected by RT- qPCR) )expression. The results showed that the cell survival rate and proliferation rate were the highest when the concentration of BMP4 was 5 ~ 20 ng · mL ~ (-1); Mvh, Fragilis, Oct-4, Dazl and Stella (P <0.05). The expression level of Nobox was the highest in the group of 40 ng · mL -1 BMP4, and the concentration of Nobox was significantly higher than that of the control group (P <0.05). The expression levels of Stra8 and Gdf9 in BMP4 group were not significantly different from those in control group (P> 0.05). The results suggest that the survival rate and proliferation rate of BMSCs and the expression of primordial germ cell-specific genes are the highest when the concentration of BMP4 is 20 ng · mL -1. BMP4 plays an important role in inducing the differentiation of mesenchymal stem cells into germ cells Important role.