EphB2-Fc融合蛋白促进实验性大脑皮层梗死后内源性神经干细胞的活化

EphB2-Fc promotes activation of endogenous neural stem cells after cerebral cortex infarction: experimental with hypertensive rats

目的探讨脑室内注入EphB2-Fc融合蛋白对大脑皮层梗死后内源性神经干细胞活化的影响。方法采用易卒中型肾血管性高血压大鼠,建立大脑中动脉闭塞模型(MCAO),并将其随机分成:(1)假手术组,(2)脑梗死组,(3)梗死+IgG-Fc组,(4)梗死+EphB2-Fc组,每组24只。MCAO术后第4天,EphB2-Fc组大鼠经侧脑室注入20μl浓度为200μg/m l的EphB2-Fc融合蛋白,IgG-Fc组大鼠注入等量等浓度的IgG-Fc。MCAO术后第1和第4周,将各组大鼠处死取脑,用免疫组化和原位杂交检测各组大鼠不同时点梗死侧EphB2及其mRNA、巢蛋白(nestin)和多聚唾液酸-神经细胞黏附分子(PSA-NCAM)表达,W estern蛋白印迹检测各组大鼠不同时点梗死侧EphB2和nestin蛋白量的变化。结果MCAO术后1周,脑梗死组梗死侧大脑皮层和侧脑室下区EphB2及其mRNA的表达低于假手术组,梗死+EphB2-Fc组高于梗死+IgG-Fc组(均P<0.05),脑梗死组与梗死+IgG-Fc组间差异无统计学意义(P>0.05);脑梗死组梗死侧侧脑室下区nestin、PSA-NCAM的表达高于假手术组,梗死+EphB2-Fc组高于梗死+IgG-Fc组(均P<0.05),并可见PSA-NCAM阳性细胞沿胼胝体向梗死灶迁移。MCAO术后4周,各组间梗死侧EphB2及其mRNA表达水平差异无统计学意义(均P>0.05),各组脑梗死大鼠梗死侧nestin、PSA-NCAM表达水平下降,各组间nestin表达水平差异无统计学意义,但脑梗死组PSA-NCAM表达水平仍高于假手术组。结论阻断内源性EphB2的活化可促进实验性大脑皮层梗死后脑室下区神经干细胞的增殖和迁移。

Objective To explore the effects of intraventricular injection of EphB2-Fc on activation of inherent neural stem cells after cerebral cortex infarction. Methods Stroke-prone renovasvular hypertension model was established in 96 SD rats by two-kidney, two-clip method. Middle cerebral artery occlusion （MCAO） model was established in 72 of these 96 stroke-prone renovascular hypertensive rats and the other 24 rats were used as sham operation group. Then the 72 rats were randomly divided into 3 equal groups： cerebral infarction group without any treatment after the MCAO, MCAO ＋ EphB2-Fc group undergoing stereotaxical infusion of EphB2-Fc at the dose of 20μl×200μg/ ml into the lateral ventricle 4 days after the distal ligation of right middle cerebral artery, and MCAO ＋ IgG-Fc group undergoing stereotaxical infusion of IgG-Fc at the dose of 20μl×200μg/ ml into the lateral ventricle4 days after the distal ligation of right middle cerebral artery. By the ends of the first and fourth weeks after the MCAO procedure 12 rats from each group were killed and their brains were taken out to undergo in situ hybridization, immunohistochemistry and Western blotting analysis in order to determine the expression of EphB2 protein and mRNA, nestin and polysialic acid-neural cell adhesion molecule （PSA-NCAM）. Results One week after the distal ligation of right middle cerebral artery, the EphB2 protein and mRNA expression levels in the ipsilateral cortex and subventricular zone （SVZ） of the cerebral infraction group were both lower than those of the sham operation group（ P 〈 0. 05 ）, such levels of the MCAO ＋ EphB2-Fc group were higher than those of the MCAO ＋ IgG-Fc group （ both P 〈 0. 05 ）, but there was no significant difference between the cerebral infraction group and IgG-Fc group（ both P 〉 0. 05 ）, and there were no differences in such levels between the cerebral infarction group and MCAO ＋ IgG-Fc group （ both P 〉 0. 05 ） ; the nestin and PSA-NCAM expression levels in the ipsilateral SVZ of the cerebral infraction group were both higher than those of the sham operation group （ both P 〈 0. 05 ）, such levels of the MCAO ＋ EphB2-Fc group were both higher than those of the MCAO ＋ IgG-Fc group （ both P 〈 0. 05 ）, and migration of PSA-NCAM positive cells to corpus callosum could be seen. Four weeks after, there were no significant differences in the expression levels of EphB2 protein and mRNA among different groups （ all P 〉 0. 05 ）, the nestin and PSA-NCAM expression levels in the ipsilateral SVZ decreased in all groups, there were no significant differences in the expression of nestin among all groups, but the PSA-NCAM expression in the ipsilateral SVZ of the cerebral infraction group was still higher than that of the sham operation group. Conclusion Disruption of EphB2 signal promotes the proliferation and migration of endogenous neural stem cells in the SVZ after cerebral cortex infarction.