骨髓间充质干细胞神经营养因子的表达及对脊髓神经元的保护作用

The expression of BDNF and NGF of bone marrow mesenchymal stem cells and protective effect for spinal cord neurons

目的:研究骨髓间充质干细胞(BMSC)表达脑源性神经营养因子(BDNF)和神经生长因子(NGF),以及BMSC条件培养液对脊髓神经元的保护作用,探讨BMSC治疗脊髓损伤的机制,为BMSC的临床应用提供依据和指导。方法:取大鼠骨髓培养BMSC,用CD44、CD45和CD71免疫细胞化学染色进行鉴定。提取BMSCmRNA,RT-PCR检测BDNF和NGF表达,ELISA检测BMSC培养液中BDNF和NGF的含量。原代取材培养大鼠脊髓神经元,10d后用不同比例的BMSC条件培养液培养24h,热休克诱导神经元凋亡,流式细胞仪检测凋亡细胞比率。结果:BMSC贴壁生长,免疫细胞化学染色CD44(+)、CD45(-)和CD71(+)。BMSC表达BDNF和NGFmRNA,BMSC培养液中含有BDNF和NGF,随培养时间的延长培养液中BDNF和NGF的含量增加。BMSC条件培养液可以减少热休克诱导的脊髓神经元凋亡的比率,并且BMSC条件培养液含量高者有更好的保护作用。结论:BMSC表达神经营养因子BDNF和NGF,对损伤脊髓神经元有保护作用。

Objective：To study the possibility of expression of BDNF （brain derived neurotrophic factor）and NGF （nerve growth factor） in bone marrow mesenchymal stem cells （BMSC） in vitro and the protective effect of BMSC conditioned medium to spinal cord neuron.Method： BMSC was cultured, the passage 3 cells were chosen to make immunocytochemical stain for CD44,CD71 and CD45. After total mRNA was extracted,the expression of BDNF and NGF were detected in BMSC.NGF and BDNF were assayed in BMSC secretory with ELISA kit. The spinal cord neurons were collected and cultured. After 10d the culture media of neuron were changed to BMSC conditioned medium for 24h. The neurons were induced to apoptosis by heat shock. The percentage of apoptosis neurons were detected with FCM. Result：BMSCs were positive of CD44 and CD71 and expressed BDNF and NGF mRNA. NGF and BDNF were found in BMSC secretory and their concentrations were increased with time. BMSC conditioned medium could decrease the rate of apoptosis of spinal cord neurons induced by heat shock.Conclusion：BMSC can express BDNF and NGF,and BMSC conditioned medium have protective effect to spinal cord neuron.