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BMP4在诱导骨髓间充质干细胞向生殖细胞分化中的作用
范静,殷慧群,汪存利,姜宏
0
(解放军第105医院生殖医学中心,合肥 230031)
摘要:
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80 ng·mL-1)的BMP4作为实验组,未添加BMP4为对照组。4 d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-1Mvh、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40 ng·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Stra8Gdf9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。
关键词:  骨形态发生蛋白4  骨髓间充质干细胞  生殖细胞  分化
DOI:10.13610/j.cnki.1672-352x.20160929.001
投稿时间:2016-06-19
基金项目:南京军区医学科技创新课题(11Z010)资助。
Differentiation of mouse bone marrow stem cells to germ cells induced by bone morphogenetic protein 4
FAN Jing,YIN Huiqun,WANG Cunli,JIANG Hong
(Reproductive Medical Center, the 105th Hospital of PLA, Hefei 230031)
Abstract:
To investigate the effect of bone morphogenetic protein 4 (BMP4) on induction of bone marrow stem cells (BSCs) to germ cells differentiation, BSCs were isolated from mouse femur and tibia, purified from other cells based on their adherent properties and cultured in the DMEM media supplemented with 10% fetal bovine serum (FBS). Passage 3 BSCs treated with different doses of BMP4(5, 10, 20, 40 and 80 ng·mL-1)were defined as experimental groups and without BMP4 as the control group. After cultivation for 4 days, the survival and proliferation rates of BSCs were measured by Trypan blue staining and MTT colorimetric assay, respectively. Simultaneously, the expression levels of germ cell-specific genes (Mvh, Oct-4, Fragilis, Nobox, Dazl, Stella, Stra8, and Gdf9) were analyzed by real-time quantitative PCR (RT-qPCR). As results, the highest cell survival and proliferation rates were obtained when the concentration of BMP4 was in the range of 5-20 ng·mL-1. Compared with the control group, a significant up-regulation of the expressions of Mvh, Fragilis, Oct-4, Dazl and Stella in BSCs under 20 ng·mL-1 BMP4 was observed (P<0.05). The expression of Nobox was up-regulated significantly in all of BMP4 groups compared with the control group (P<0.05) and the highest expression was in the group of 40 ng·mL-1 BMP4. The expression levels of Stra8 and Gdf9 at all of BMP4 concentrations showed no significant differences with the control group. In conclusion, 20 ng·mL-1 BMP4 may be the optimal concentration in promoting the cell survival and proliferation as well as the expression of the germ cell-specific gene, and BMP4 may play an important role in facilitating differentiation of BSCs to germ cells.
Key words:  bone morphogenetic proteins 4  bone marrow mesenchymal stem cell  germ cell  differentiation

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