整合素β4调控脑血管发育

Integrinβ4 Regulates Cerebral Vascular Development in Zebrafish

【目的】探讨整合素β4(ITGB 4)对脑血管生长发育的调控作用。【方法】通过逆转录PCR(RT-PCR)检测靶向itgb 4 mRNA序列的吗啉环(MO)在斑马鱼胚胎中的敲低效果。利用体视显微镜和激光共聚焦显微镜(laser scanning confocal microscope,LSCM)观察抑制itgb 4表达后,斑马鱼胚胎脑部血管的发育变化。通过在斑马鱼胚胎中注射4,6-联脒-2-苯基吲哚(DAPI)染料检测血脑屏障(BBB)发育的完整性。使用小干扰RNA(small interfering RNA,siRNA)在永生化人脑微血管内皮细胞(hCMEC/D 3)中敲低ITGB 4,用蛋白免疫印迹(WB)检测其敲低效果。通过活细胞成像分析仪(IncuCyte®)、基质胶(Matrigel®matrix)成管实验和transwell迁移实验分别检测ITGB 4在hCMEC/D 3中调控细胞增殖、成管和迁移的作用。应用实时荧光定量PCR(qRT-PCR)检测敲低itgb 4后Wnt/β-catenin信号通路下游靶基因表达变化。【结果】在斑马鱼胚胎中敲降itgb 4引起脑水肿、脑部出血、血脑屏障形成障碍以及后脑血管数量减少等相关表型。细胞实验结果显示,在hCMEC/D 3细胞中抑制ITGB 4的表达导致细胞增殖减缓,管腔形成减少及迁移能力减弱。此外,qRT-PCR结果表明,在斑马鱼胚胎中敲低itgb 4抑制Wnt/β-catenin通路下游靶基因的表达。最后,在hCMEC/D 3细胞中敲低ITGB 4后,通过添加Wnt/β-catenin通路激活剂(BML-284)能明显挽回迁移减少的表型。【结论】ITGB 4通过调节脑血管内皮细胞的增殖、迁移和成管等功能促进脑血管发育并参与调控血脑屏障的形成。

【Objective】To explore the effect of integrinβ4(ITGB 4)on cerebrovascular development.【Methods】We confirmed the knock down effect of morpholino(MO)targeting itgb 4 mRNA sequence on zebrafish embryos by using RT PCR.Upon the knockdown effect of itgb 4 in zebrafish embryos by MO,we observed the phenotypes of cerebrovascular de⁃velopment via laser scanning confocal microscope and stereomicroscope.To examine the integrity of blood-brain barrier(BBB),we injected the DAPI to the zebrafish embryos.To detect the functional changes of cerebrovascular endothelial cells,we inhibited the expression of ITGB 4 by siRNA and confirmed the knockdown effect by western blot(WB).To in⁃spect the role of ITGB 4 in brain microvascular endothelial cell(hCMEC/D 3),we performed cell proliferation assay,tube formation assay and cell migration assay on ITGB 4 knock-down cells.We used qRT-PCR to detect the change of the down⁃stream target genes in the Wnt/β-catenin pathway upon the knockdown effect of itgb 4.【Results】Itgb 4 knocked down by MO resulted in dramatic defects in the zebrafish embryos,including brain edema,brain hemorrhage,blood-brain barrier dysfunction and reduction of the number of the central arteries(CtAs)in hindbrain.Downregulation of ITGB 4 protein ex⁃pression in hCMEC/D 3 disrupted the cell function,including the suppression of cellular proliferation,the reduction of the tube formation and the inhibition of cellular migration.In addition,the downstream target genes in the Wnt/β-catenin sig⁃nal pathway were suppressed since the downregulation of itgb 4 expression in zebrafish embryos.Finally,the reduction of the cells migration after knocking down the ITGB 4 could be rescued when we add the activator(BML-284)of the Wnt/β-catenin signal pathway to the hCMEC/D 3 cells.【Conclusions】ITGB 4 promotes the development of cerebrovascular by regu⁃lating cellular proliferation,migration and tube formation of cerebral vascular endothelial cells,and plays an important role in regulating the formation of blood-brain barrier.