VEGF介导PI3/K信号通路保护脊髓运动神经元的实验研究

The effects of VEGF on glutamate-induced impairment of spinal anterior-horn neurons

目的建立脊髓前角运动神经元兴奋性氨基酸损伤的模型,以研究不同浓度血管内皮生长因子(Vascular endothelial growth factor,VEGF)干预及磷脂酰肌醇3-激酶(Phosphatidyl-inositol3-kinase,PI3/K)抑制剂对其活力及PI3/K表达的影响。方法体外培养新生SD大鼠脊髓运动神经元,并建立对照组、VEGF组、谷氨酸(Glutamate,Glu)组及PI3/K组,通过MTT法检测各组运动神经元活力,观察各组大鼠脊髓运动神经元的数量,采用免疫荧光方法及酶标记免疫吸附测定法检测PI3/K的表达水平。结果筛选出适宜建立兴奋性氨基酸损伤模型的Glu浓度为5 umol/ml。在Glu干预下VEGF0.1 ug/ml组的细胞活力较VEGF1 ug/ml组显著降低(P<0.05);VEGF1 ug/ml加PI3/K组的细胞活力较VEGF1 ug/ml组显著降低(P<0.05);通过免疫荧光法及ELISA法检测运动神经元的PI3/K表达时也有相似的发现。结论 VEGF可改善由Glu所造成的脊髓运动神经元损伤,增加损伤神经元PI3/K的表达,且在一定浓度范围内存在量效关系。通过LY294002抑制VEGF增加细胞PI3/K表达的作用,推测VEGF对运动神经元的保护作用可能部分通过PI3/K信号通路完成。

Objective To study the effects of different concentrations of vascular endothelial growth factor（VEGF） on glutamine-induced impairment of spinal outerior-horn neurons,and its phosphatidylinositol 3-kinase（PI3/K） expression. Methods Spinal motor neurons were dissociated from Sprague-Dawley rats,cultured in vitro,and then were exposed to culture media only（control group）,VEGF（VEGF group）,VEGF plus glutamate（Glutamate group）,VEGF plus PI3/K inhibitor（Inhibitor group）,respectively.Viability of motor neurons was assessed by MTT test,while PI3/K expression was observed by immunofluorescence and ELISA. Results The optimal concentration of glutamate to establish the model was 5umol/ml.It was also found VEGF could ameliorate glutamate-induced impairment of cell viability in a dose-dependent manner（P0.05）.However,PI3/K inhibitor LY294002 could reduce the effects of VEGF（P0.05）.Similar findings were also observed in PI3/K expression among different groups by immunofluorescence and ELISA detection. Conclusions The cultured motor neurons in vitro showed reduced viability with increasing concentration of glutamate treatment.Low and moderate concentration of VEGF ameliorated the viability of glutamate-treated spinal motor neurons and increased PI3/K expression in a dose-dependent manner.LY294002,a PI3/K inhibitor,decreased the viability of VEGF and glutamate-treated cultured motor neurons significantly,suggesting the involvement of PI3/K pathway in the protective effects of VEGF on glutamate-induced impairment of spinal motor neurons.