目的：探讨 TrkBBDNF 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+BDNF 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。
Abstract
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.
关键词
神经母细胞瘤 /
TrkB-BDNF /
LY294002 /
血管内皮生长因子 /
SH-SY5Y 细胞
Key words
Neuroblastoma /
TrkB-BDNF /
LY294002 /
Vascular endothelial growth factor /
SH-SY5Y cell
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