【作者】 王志坚；

【导师】 郑关毅；

【作者基本信息】 福建医科大学 ， 中西医结合临床， 2011， 硕士

【摘要】 目的:探讨毛冬青甲素(Ilexonin,IA)对大鼠脑缺血再灌注后VEGF和VEGFR2的表达及神经元再生的影响的可能作用机制。方法:线栓左侧大脑中动脉阻塞(middle cerebral artery occlusion,MCAO)制成SD大鼠脑缺血模型,随机分成:正常组、假手术组、模型组、IA治疗组(分IA71、142、285μmol·kg- 1三个亚组)等4组。采用Longa评分法进行大鼠神经功能评分; TTC染色法观察脑缺血再灌注模型组和治疗组缺血灶情况;免疫组化检测缺血再灌注后不同时段各组缺血周边区VEGF、VEGFR2阳性细胞数的变化;免疫荧光检测缺血再灌注后不同时段各组脑侧室室管膜下区(SVZ)神经元再生标记物Nestin阳性细胞数的变化;免疫印迹法检测缺血再灌注后不同时段各组缺血周边区VEGF、VEGFR2及脑侧室室管膜下区(SVZ)神经元再生标记物Nestin蛋白的表达情况。并比较不同剂量(IA71、142、285μmol·kg- 1)的IA干预对大鼠神经功能缺损评分,缺血区周边组织VEGF、VEGFR2及SVZ的Nestin蛋白表达和阳性细胞数的影响。结果:脑缺血再灌注后3d,模型大鼠神经功能缺损评分较高(P<0.05 VS 14d、28d);VEGF、VEGFR2、Nestin阳性细胞在正常组及假手术组散在见到,模型组7d达到高峰,随时间延长其表达逐渐减少;VEGF、VEGFR2、Nestin蛋白于正常组及假手术组表达较少,模型组自第3d有一定量的表达,第7天表达最多,之后逐渐减少,至28天仍有表达。模型组7d与相应模型组其它时段比较P<0.05。经不同剂量IA71、142、285μmol·kg- 1干预后,大鼠神经功能缺损评分有明显下降;缺血周边区VEGF、VEGFR2及SVZNestin蛋白表达和阳性细胞数明显增多,与模型组相应时间点比较显著性差异(P<0.05)。以285μmol·kg- 1作用为著。结论:1、脑缺血再灌注损伤可诱导VEGF、VEGFR2的早期表达(至少14d), VEGF、VEGFR2的早期应激表达可能与及时挽救缺血周边组织神经元的存活和再生进而促进神经功能恢复有关。2、毛冬青甲素可以促进缺血周边组织VEGF、VEGFR2表达(至少14d),其机制可能通过上调VEGF、VEGFR2表达促进神经元的存活和再生进而发挥其神经保护作用。更多还原

【Abstract】 OBJECTIVE To explore the effect of IlexoninA on expression of VEGF, VEGFR2 and neurogenesis and its possible mechanism after cerebral ischemia- reperfusion in Rats.METHODS The model of Middle cerebral artery occlusion(MCAO) was established to transient focal ischemia by placement of an intraluminal filament at the origin of left middle cerebral artery. The rats were divided randomly into: control, sham operation, model and IlexoninA (IA71、142、285μmol·kg- 1) treatment groups. Neuronogical behavior was evaluated by Longa’s scoring method. TTC staining was applied to observe cerebral ischemia in MCAO model group and IlexoninA treatment groups. Immunohistochemistry were used to observe the expression of VEGF, VEGFR2 positive cells in the ischemic brain tissue in theat different time of ischemia and reperfusion. Immunofluorescence were used to observe the expression of Nestin (neurogenesis Marker)positive cells in the SVZ in theat different time of ischemia and reperfusion.Western bloting were used to observe the expression of VEGF, VEGFR2 protein in the ischemic brain tissue and Nestin protein(neurogenesis Marker) in the subventricular zone in theat different time of ischemia and reperfusion.The effects of IA intervention (71、142、285μmol·kg- 1) on neurological deficit score and the expression of VEGF, VEGFR2 protein in ischemia surrounding areas and Nestin protein in ischemia SVZ were mainly quantified.And the effects of IA intervention (71、142、285μmol·kg- 1) on the positive cells of VEGF, VEGFR2 in ischemia surrounding areas and Nestin in ischemia SVZ were mainly quantified.RESULTS Neurological deficits of model were more at 3d(P<0.05 compared to 14d and 28d).In control and sham groups, there were scattered VEGF, VEGFR2 positive cells (mainly in the cortex), and the number of VEGF, VEGFR2 positive cells were peaked at 7d in model group. With the prolongation of reperfusion, the expression of VEGF, VEGFR2 decreased.VEGF, VEGFR2 protein in the control group and sham group was much less than model group.In model group, there was a certain amount at 3d after reperfusion, reached to peak at 7d, then reduced gradually, to 28d was still expression. Similarly, Nestin protein reached its peak at 7d after reperfusion, and then decrcased gradually, compared to other model times (P<0.05). After the intervention of IA71、142、285μmol·kg- 1, the neurological deficit score decreased significantly, and the expression of VEGF, VEGFR2 protein in ischemia surrounding areas and Nestin in ischemia SVZ were increased comparing to model group at corresponding time points (P<0.05), and the positive cells of VEGF, VEGFR2 in ischemia surrounding areas and Nestin in ischemia SVZ were increased comparing to model group at corresponding time points (P<0.05), with pronounced effct at dose of IA285μmol·kg- 1.CONCLUSION1.Cerebral ischemia reperfusion injury induced the expression of VEGF, VEGFR2 in the early time(at least 14d). It is possible that the early expression of VEGF, VEGFR2 were involved to promote the survival and remodeling of peripheral neurons ,with promoting the recovery of neurological function.2. IlexoninA can promote the the expression of VEGF, VEGFR2 in the early time(at least 14d).It may be passed through upregulation of VEGF, VEGFR2 to promote the repair and regeneration of neurons to play its role of neuroprotective.更多还原