中国小儿急救医学
中國小兒急救醫學
중국소인급구의학
CHINESE PEDIATRIC EMERGENCY MEDICINE
2013年
4期
398-402
,共5页
刘建英%高惠敏%李爱敏%蔡维艳%初清
劉建英%高惠敏%李愛敏%蔡維豔%初清
류건영%고혜민%리애민%채유염%초청
神经母细胞瘤%TrkB-BDNF信号传导通路%K252a%LY294002%血管内皮生长因子%基质金属蛋白酶-9
神經母細胞瘤%TrkB-BDNF信號傳導通路%K252a%LY294002%血管內皮生長因子%基質金屬蛋白酶-9
신경모세포류%TrkB-BDNF신호전도통로%K252a%LY294002%혈관내피생장인자%기질금속단백매-9
Neuroblastoma%TrkB-BDNF signal pathway%K252a%LY294002%Vascular endothelial growth factor%Matrix metalloproteinases-9
目的 探讨酪氨酸激酶受体B(tyrosine kinase receptor B,TrkB)-脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)信号传导通路对神经母细胞瘤(neuroblastoma,NB)细胞SH-SY5Y分泌血管内皮生长因子(vascular endothelial growth factor,VEGF)及基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)的影响.方法 用全反式维甲酸(all-trans retinoic acid,ATRA)诱导SH-SY5Y细胞表达TrkB,加入外源性BDNF,从而激活TrkB-BDNF信号传导通路及其3条下游信号通路.再用特异性酪氨酸激酶抑制剂K252a阻断TrkB-BDNF信号传导通路；用磷脂酰肌醇-3激酶(phosphatidylinositol 3-hydrox y kinase,PBK)抑制剂LY294002、磷脂酶C抑制剂U73122、丝裂原活化蛋白激酶抑制剂U0126分别阻断TrkB-BDNF的3条相应下游信号传导通路.采用酶联免疫吸附法检测细胞培养上清液中VEGF及MMP-9含量.结果 ATRA+ BDNF组VEGF[(485.89±109.99) pg/ml]及MMP-9[(15.73±1.72) pg/ml]含量明显高于对照组及ATRA组(P ＜0.05)；ATRA+ BDNF+ K252a组VEGF[(272.42±86.33) pg/ml]及MMP-9[(5.25±1.44) pg/ml]含量明显低于ATRA+ BDNF组(P＜0.05),与对照组及ATRA组比较差异无统计学意义(P＞0.05)；ATRA+ BDNF+ LY294002组VEGF[(314.12±24.68) pg/ml]及MMP-9[(4.91±1.08) pg/ml]含量明显低于ATRA+ BDNF组(P＜0.05),与对照组及ATRA组比较差异无统计学意义(P ＞0.05)；ATRA+ BDNF+ U73122组VEGF[(444.08±64.49) pg/ml]及MMP-9[(13.28±3.38) pg/ml]含量与ATRA+ BDNF组比较差异无统计学意义(P＞0.05).ATRA+ BDNF+ U0126组VEGF[(429.97±19.95) pg/ml]及MMP-9[(13.96±4.45) pg/ml]含量与ATRA+ BDNF组比较差异无统计学意义(P＞0.05).结论 激活TrkB-BDNF信号传导通路可促进NB细胞合成、分泌VEGF及MMP-9.TrkB-BDNF信号传导通路可能通过进一步激活其下游PI3 K/Akt通路来促进VEGF及MMP-9的合成及分泌,用K252a阻断TrkB-BDNF信号通路、LY294002阻断其下游PI3K通路均可有效抑制NB细胞合成及分泌VEGF及MMP-9.
目的 探討酪氨痠激酶受體B(tyrosine kinase receptor B,TrkB)-腦源性神經營養因子(brain-derived neurotrophic factor,BDNF)信號傳導通路對神經母細胞瘤(neuroblastoma,NB)細胞SH-SY5Y分泌血管內皮生長因子(vascular endothelial growth factor,VEGF)及基質金屬蛋白酶-9(matrix metalloproteinase-9,MMP-9)的影響.方法 用全反式維甲痠(all-trans retinoic acid,ATRA)誘導SH-SY5Y細胞錶達TrkB,加入外源性BDNF,從而激活TrkB-BDNF信號傳導通路及其3條下遊信號通路.再用特異性酪氨痠激酶抑製劑K252a阻斷TrkB-BDNF信號傳導通路；用燐脂酰肌醇-3激酶(phosphatidylinositol 3-hydrox y kinase,PBK)抑製劑LY294002、燐脂酶C抑製劑U73122、絲裂原活化蛋白激酶抑製劑U0126分彆阻斷TrkB-BDNF的3條相應下遊信號傳導通路.採用酶聯免疫吸附法檢測細胞培養上清液中VEGF及MMP-9含量.結果 ATRA+ BDNF組VEGF[(485.89±109.99) pg/ml]及MMP-9[(15.73±1.72) pg/ml]含量明顯高于對照組及ATRA組(P ＜0.05)；ATRA+ BDNF+ K252a組VEGF[(272.42±86.33) pg/ml]及MMP-9[(5.25±1.44) pg/ml]含量明顯低于ATRA+ BDNF組(P＜0.05),與對照組及ATRA組比較差異無統計學意義(P＞0.05)；ATRA+ BDNF+ LY294002組VEGF[(314.12±24.68) pg/ml]及MMP-9[(4.91±1.08) pg/ml]含量明顯低于ATRA+ BDNF組(P＜0.05),與對照組及ATRA組比較差異無統計學意義(P ＞0.05)；ATRA+ BDNF+ U73122組VEGF[(444.08±64.49) pg/ml]及MMP-9[(13.28±3.38) pg/ml]含量與ATRA+ BDNF組比較差異無統計學意義(P＞0.05).ATRA+ BDNF+ U0126組VEGF[(429.97±19.95) pg/ml]及MMP-9[(13.96±4.45) pg/ml]含量與ATRA+ BDNF組比較差異無統計學意義(P＞0.05).結論 激活TrkB-BDNF信號傳導通路可促進NB細胞閤成、分泌VEGF及MMP-9.TrkB-BDNF信號傳導通路可能通過進一步激活其下遊PI3 K/Akt通路來促進VEGF及MMP-9的閤成及分泌,用K252a阻斷TrkB-BDNF信號通路、LY294002阻斷其下遊PI3K通路均可有效抑製NB細胞閤成及分泌VEGF及MMP-9.
목적 탐토락안산격매수체B(tyrosine kinase receptor B,TrkB)-뇌원성신경영양인자(brain-derived neurotrophic factor,BDNF)신호전도통로대신경모세포류(neuroblastoma,NB)세포SH-SY5Y분비혈관내피생장인자(vascular endothelial growth factor,VEGF)급기질금속단백매-9(matrix metalloproteinase-9,MMP-9)적영향.방법 용전반식유갑산(all-trans retinoic acid,ATRA)유도SH-SY5Y세포표체TrkB,가입외원성BDNF,종이격활TrkB-BDNF신호전도통로급기3조하유신호통로.재용특이성락안산격매억제제K252a조단TrkB-BDNF신호전도통로；용린지선기순-3격매(phosphatidylinositol 3-hydrox y kinase,PBK)억제제LY294002、린지매C억제제U73122、사렬원활화단백격매억제제U0126분별조단TrkB-BDNF적3조상응하유신호전도통로.채용매련면역흡부법검측세포배양상청액중VEGF급MMP-9함량.결과 ATRA+ BDNF조VEGF[(485.89±109.99) pg/ml]급MMP-9[(15.73±1.72) pg/ml]함량명현고우대조조급ATRA조(P ＜0.05)；ATRA+ BDNF+ K252a조VEGF[(272.42±86.33) pg/ml]급MMP-9[(5.25±1.44) pg/ml]함량명현저우ATRA+ BDNF조(P＜0.05),여대조조급ATRA조비교차이무통계학의의(P＞0.05)；ATRA+ BDNF+ LY294002조VEGF[(314.12±24.68) pg/ml]급MMP-9[(4.91±1.08) pg/ml]함량명현저우ATRA+ BDNF조(P＜0.05),여대조조급ATRA조비교차이무통계학의의(P ＞0.05)；ATRA+ BDNF+ U73122조VEGF[(444.08±64.49) pg/ml]급MMP-9[(13.28±3.38) pg/ml]함량여ATRA+ BDNF조비교차이무통계학의의(P＞0.05).ATRA+ BDNF+ U0126조VEGF[(429.97±19.95) pg/ml]급MMP-9[(13.96±4.45) pg/ml]함량여ATRA+ BDNF조비교차이무통계학의의(P＞0.05).결론 격활TrkB-BDNF신호전도통로가촉진NB세포합성、분비VEGF급MMP-9.TrkB-BDNF신호전도통로가능통과진일보격활기하유PI3 K/Akt통로래촉진VEGF급MMP-9적합성급분비,용K252a조단TrkB-BDNF신호통로、LY294002조단기하유PI3K통로균가유효억제NB세포합성급분비VEGF급MMP-9.
Objective To study the effects of tyrosine kinase receptor B-brain-derived neurotrophic factor (TrkB-BDNF) signal pathway on the secretion of vascular endothelial growth factor (VEGF) and matrix metalloproteinases-9(MMP-9) of neuroblastoma.Methods We used all-trans retinoic acid (ATRA) to induce the high expression of TrkB in the SH-SY5Y cell line,and then added the ectogenid BDNF to activate the TrkB-BDNF and its three downstream signal pathways.TrkB-BDNF signal pathway was inhibited by specific tyrosine kinase inhibitor K252a.The three downstream signal pathway was respectively inhibited by LY294002 (the phosphatidylinositol 3-hydroxy kinase (PI3 K) pathway inhibitor)、U73122 (the phospholipase C pathway inhibitor) 、U0126(the mitogen activated protein kinase pathway inhibitor).Enzyme linked immunosorbent assay was used to detect the concentration of VEGF and MMP-9 protein in the SY5Y cell culture supernatants.Results VEGF [(485.89 ± 109.99) pg/ml] and MMP-9 [(15.73 ± 1.72) pg/ml] protein levels in neuroblastoma cells cultured in serum-free media in the group of ATRA + BDNF were significantly higher than that of the control group and ATRA alone group(P ＜0.05).VEGF [(272.42 ±86.33) pg/ml]and MMP-9 [(5.25 ± 1.44) pg/ml] protein levels in the group of ATRA + BDNF + K252a were significantly lower than those of the ATRA + BDNF group(P ＜ 0.05) and had no significant difference compared with the control group and the ATRA alone group(P ＞0.05).VEGF [(314.12 ±24.68) pg/ml] and MMP-9 [(4.91 ± 1.08) pg/ml] protein levels in the group of ATRA + BDNF + LY294002 were significantly lower than those of the ATRA + BDNF group(P ＜ 0.05) and had no significant difference compared with the control group and the ATRA alone group(P ＞0.05).VEGF [(444.08 ±64.49) pg/ml] and MMP-9 [(13.28 ±3.38) pg/ml] protein levels in neuroblastoma cells cultured in serum-free media in the group of ATRA +BDNF + U73122 had no significant difference compared with the ATRA + BDNF group(P ＞ 0.05).VEGF [(429.97 ± 19.95) pg/ml] and MMP-9 [(13.96 ± 4.45) pg/ml] protein levels in neuroblastoma cells cultured in serum-free media in the group of ATRA + BDNF + U0126 had no significant difference compared with the ATRA + BDNF group(P ＞ 0.05).Conclusion Activation of TrkB-BDNF signal pathway can increase the synthesis and secretion of VEGF and MMP-9 in human neuroblastoma cells.TrkB-BDNF signal pathway may be through activating its downstream PI3K pathway to increase the synthesis and secretion of VEGF and MMP-9 in human neuroblastoma cells.The synthesis and secretion of VEGF and MMP-9 can be inhibited by blocking the TrkB-BDNF signal pathway with K252a or blocking its downstream signal pathway PI3 K with LY294002.
