VEGF对缺血性脑损伤的作用及作用机制

Previous studies showed that vascular endothelial growth facor (VEGF), an important neurothophic factor, can be expressed by neuron, astrocyte and microglia in the adult brain. Cerebral ischemia induces the expression of neuronal VEGF and exogenous VEGF exerts its neuroprotective role via reducing the apoptosis of vascular endothelium. However, it still unclear of the role and probable mechanism of the induced endogenous VEGF.In the present study, in situ hybridization and double fluorescent staining combined with confocal laser scanning microscopy were used to analyze the expression and cellular localization of endogenous VEGF. It showed that cerebral ischemia could significantly induced VEGF and its mRNA expression in rat brain and VEGF positice staining was mainly existed in neuron and colocalized with excision repair cross-complementing group 6 (ERCC6) mRNA. VEGF antibody and antisense infusion could significantly reduced the endogennous VEGF induction and enlarged the infarct volume. Moreover, it also increased the number of DNA damaged cells and lessened the expression of ERCC6 mRNA. These results indicated the ischemia-induced endogenous neuronal VEGF plays neuroprotective role associating with the expression of ERCC6 mRNA..On the other hand, the outward delayed rectifer potassium current plays an important role in detemining the fate of ischemic neurons. Double fluorescent staining combined with confocal laser scanning microscopy, whole-cell patch clamp, Immunoprecipitation and western blot analysis were used to show Flt-1 and Flk-1 expressed in the neurons with separate time-dependent manners and regional distribution in neonatal and adult rat brain; VEGF could decreased the outward delayed rectifer potassium current in VEGF receptor expressed hippocampal neurons. This effect was related to stimulate the activities of its receptor's tyrosine kinase. Futher studies showed kv1.2, an isoform of delayed-rectifier potassium channel, mainly located in neurons and was up-regulated in the in vitro and in vivo ischemia models; VEGF increased the tyrosine phosphorylation of Kv1.2 via activating the tyrosine kinases and PI3-Ksignaling transduction pathway in ischemic cells, which resulted in an inhibition of outward delayed rectifer potassium current. These results suggest the VEGF-decreased outward delayed rectifer potassium current is relevant to the VEGF receptors and the phosphorylation of Kv1.2.In all, the ischemia-induced endogenous VEGF plays neuroprotective role via adjusting the expression of ERCC6 mRNA and the level of outward delayed rectifer potassium.

本研究在已建立的整体及离体的神经细胞损伤模型上，应用分子生物学，免疫学及经典药理学手段，通过改变血管内皮生长因子(VEGF)的生物转化过程及含量，观察VEGF对神经细胞损伤的影响，在此基础上了解VEGF对神经细胞损伤后突触可塑性的影响，进一步用电生理方法研究VEGF对离子通道的影响，以此探讨VEGF对神经细胞的损伤后的作用及其作用机制.