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目的探讨血管内皮生长因子(VEGF)对6羟基多巴胺(6OHDA)诱导的PC12细胞变性损伤的保护及机制。方法用PC12细胞制作帕金森病细胞模型,采用四甲基偶氮唑蓝法检测暴露于梯度浓度VEGF165后细胞的活性;流式细胞术检测对照组、损伤组(单加6OHDA)、VEGF组(单加VEGF165)和VEGF保护组(同时加6OHDA和VEGF165)细胞凋亡率(每组5个样本);免疫印迹法检测PC12细胞VEGF165及其受体fms样酪氨酸激酶1(Flt1)、胎肝激酶1(Flk1)、神经纤毛蛋白1(Nrp1)的表达,并观察VEGF165及其受体中和抗体对VEGF165作用的影响。结果随着VEGF165的浓度增加到50～100ng/ml时,PC12细胞吸光度由0.14±0.06逐步上升至0.35±0.08(P<0.01),随后又稍下降;细胞凋亡率VEGF保护组(29.65%±6.63%)低于损伤组(57.10%±5.66%),但高于VEGF组(3.67%±1.51%)和对照组(4.76%±1.06%,P<0.01);免疫印迹实验检测到PC12细胞有VEGF165及Flt1和Nrp1的表达,而未检测到Flk1;抗VEGF165和抗Nrp1中和抗体可阻断VEGF165的作用,而抗Flt1抗体无作用。结论VEGF165可保护PC12细胞因6OHDA造成的损伤,Nrp1受体在其中发挥重要作用。
Objective To investigate the protective effect of vascular endothelial growth factor (VEGF) on the degeneration of PC12 cells induced by 6-hydroxydopamine (6OHDA) and its mechanism. Methods The PC12 cells were used to make the cell model of Parkinson’s disease. The cell viability was detected by MTT assay after exposure to gradient concentration of VEGF165. Flow cytometry was used to detect the expression of VEGF165 in the control group, injury group (6OHDA alone) and VEGF group (5 samples in each group), VEGF165 group and VEGF protection group (plus 6OHDA and VEGF165). The expression of VEGF165 and its receptor fms-like tyrosine kinase 1 (Flt1) in PC12 cells were detected by immunoblotting, The expression of Flk1 and Nrp1 was observed and the effect of VEGF165 and its receptor neutralizing antibody on the effect of VEGF165 was also observed. Results As the concentration of VEGF165 increased to 50-100 ng / ml, the absorbance of PC12 cells gradually increased from 0.14 ± 0.06 to 0.35 ± 0.08 (P <0.01), and then decreased slightly. The apoptotic rate of VEGF protective group (29.65% ± 6.63%) were lower than those in the injured group (57.10% ± 5.66%), but higher than those in the VEGF group (3.67% ± 1.51%) and the control group (4.76% ± 1.06%, P <0.01) VEGF165 and Flt1 and Nrp1, but no detectable Flk1; anti-VEGF165 and anti-Nrp1 neutralizing antibodies could block the effect of VEGF165, whereas anti-Flt1 antibody had no effect. Conclusion VEGF165 protects PC12 cells against 6OHDA injury and Nrp1 receptor plays an important role.