TrkB-BDNF信号通路对神经母细胞瘤细胞分泌血管内皮生长因子的影响

Effects of TrkB-BDNF signal pathway on synthesis and secretion of vascular endothelial growth factor in human neuroblastoma cells

目的探讨TrkB-BDNF信号通路对神经母细胞瘤(NB)细胞SH-SY5Y分泌血管内皮生长因子(VEGF)的影响。方法 Western blot方法检测全反式维甲酸(ATRA)诱导前后SY5Y细胞TrkB蛋白表达及脑源性神经营养因子(BDNF)刺激后SY5Y细胞磷酸化-TrkB(p-TrkB)蛋白表达;ELISA技术检测ATRA、BDNF、特异性酪氨酸激酶抑制剂K252a及PI3K抑制剂LY294002处理后SY5Y细胞培养上清中VEGF含量。结果 ATRA诱导前,SY5Y细胞中未检测到TrkB蛋白表达;1,10,100 nM/L ATRA处理后,SY5Y细胞中可检测到TrkB蛋白表达,且TrkB蛋白表达水平随ATRA浓度增加逐渐升高,差异有统计学意义。10 nM/L ATRA单独处理组未检测到p-TrkB表达,ATRA+BDNF组可检测p-TrkB蛋白表达。ATRA+BDNF组的VEGF含量明显高于对照组及ATRA组(P<0.01);ATRA+K252a+BDNF组VEGF含量明显低于ATRA+BDNF组(P<0.05);ATRA+LY294002+BD-NF组VEGF含量亦明显低于ATRA+BDNF组(P<0.01)。结论激活TrkB-BDNF信号通路可促进NB细胞合成、分泌VEGF;用K252a阻断TrkB-BDNF信号通路或用LY294002阻断TrkB-BDNF信号下游通路PI3K/Akt均可有效抑制NB细胞合成、分泌VEGF。

Objective To study the effects of TrkB-BDNF signal pathway on the synthesis and secretion of vascular endothelial growth factor（VEGF） in human neuroblastoma cells（NB）. Methods TrkB protein expression in SY5Y cells before and after all-trans-retinoicacid（ATRA） treatment was detected by Western blot.p-TrkB protein expression in SY5Y cells before and after the treatment of ATRA along with BDNF was also detected by Western blot.VEGF concentrations in the SY5Y cell culture supernatants were measured using ELISA after the treatment with ATRA,BDNF,tyrosine kinase inhibitor K252a and PI3k inhibitor LY294002. Results TrkB protein was undetectable in SY5Y cells before ATRA treatment.After the treatment of 1,10 and 100 nM/L ATRA for five days,TrkB protein was expressed in SY5Y cells and the TrkB protein level increased with the increasing ATRA concentration.p-TrkB protein was not expressed in SY5Y cells treated only with 10 nM/L ATRA,but it was detectable after the treatment of ATRA along with BDNF.VEGF concentrations in the group treated with ATRA＋BDNF were significantly higher than those in the untreated control and the ATRA alone treatment groups（P〈0.01）.VEGF concentrations in the K252a pretreated ATRA＋BDNF group were significantly lower than those in the group treated with ATRA＋BDNF（P〈0.05）.VEGF concentrations in the LY294002 treatment group（ATRA＋LY294002＋BDNF group） were also significantly lower than those in the group treated with ATRA＋BDNF（P〈0.01）. Conclusions Activation of TrkB-BDNF signal pathway may increase the synthesis and secretion of VEGF in human NB cells.The synthesis and secretion of VEGF can be inhibited by blocking TrkB-BDNF signal pathway with K252a or blocking the TrkB-BDNF downstream signal pathway PI3K/Akt with LY294002.