脑源性神经营养因子对无血清培养的神经元保护机制的研究

Protection mechanism of brain derived neurotrophic factor on neuron-like cells under serum free condition

目的探讨在无血清培养条件下脑源性神经营养因子(BDNF)对神经元样细胞存活的影响及其作用机制。方法在无血清条件下培养具有BDNF受体TrkB表达的神经母细胞瘤细胞SY5Y-TrkB,在培养液中单独加入BDNF,或联合加入磷脂酰肌醇3-激酶(PI3K)抑制剂/丝裂原活化蛋白激酶(MAPK)抑制剂进行细胞培养,利用细胞活性测定法(MTS)检测活细胞活性。结果与在10%胎牛血清(FBS)培养液中培养的细胞相比,无血清条件下培养24、48 72 h后,SY5Y-TrkB细胞的存活率分别为51%、38%、25%。若细胞在无血清条件下培养24 h,无处理因素的细胞存活率设定为100%,给予BDNF后的细胞存活率为154%;用10μmol/L PI3K抑制剂LY294002预处理1 h,再给予BDNF后的细胞存活率为100%;而应用80μmol/LMAPK抑制剂PD98059预处理1 h,再给予BDNF后的细胞存活率为158%。结论 BDNF通过PI3K信号通路保护SY5Y-TrkB细胞免受无血清培养引起的细胞死亡。

Objective To study the effect of brain derived neurotrophic factor （BDNF） on neuron-like cell survival under serum free condition and the related mechanism. Methods Tropomyosin receptor kinase （TrkB） expressing neuroblastoma cells SY5Y-TrkB were cultured in serum free media, treated with BDNF, or with a combination of BDNF and phosphatidylinositol 3-kinase （P13K） inhibitor or mitogen activated protein kinase （MAPK） inhibitor. Cell survival was detected by MTS assay. Results Compared to the cells cultured in 10% FBS medium, the survival rate of SY5Y-TrkB cells in serum free medium decreased to 51% by 24 h, 38% by 48 h, and 25% by 72 h. SY5Y-TrkB cells were cultured in serum-free medium for 24 hl compared to control cells whose survival rate was set as 100%, adding BDNF to the medium increased cell survival rate to 154%. Pretreatment of the cells with PI3K inhibitor LY294002 （10 μmol/L） for 1 h before BDNF resulted in a cell survival rate of 100% ; while pretreatment of cells with MAPK inhibitor PD98059 （80 μmol/L） for 1 h before BDNF resulted in a cell survival rate of 158%. Conclusion BDNF can protect SY5Y-TrkB cells from serum starvation-induced cell death through PI3K pathway. （Shanghai Med J, 2011, 34= 375-378）