Ang1-Akt途径对大鼠体外血-脊髓屏障功能的调节

Ang-Akt Pathway Modulates the Function of Blood-Spinal Cord Barrier in Rat

检测血管生成素1(angiopoietin1,Ang1)对体外培养脊髓微血管内皮细胞(spinal cord microvascular endothelial cell,SCMEC)屏障功能的调节机制。体外分离培养大鼠SCMEC及胶质细胞,通过双室培养系统建立体外血-脊髓屏障(blood-spinal cord barrier,BSCB)模型;分别用Ang1及Ang1+wortmannin(Akt抑制剂)处理培养细胞,Western-blot检测Akt及连接蛋白质的表达;放射自显影检测Akt激酶活性变化;测定跨内皮电阻(TEER)反映各组细胞屏障功能;细胞免疫荧光检测连接蛋白在细胞接触面的分布变化;免疫共沉淀实验分析连接蛋白之间的相互作用改变。结果显示,Ang1处理组细胞Akt活性及TEER值均有明显升高,Ang1处理后ZO-1及VE-cadherin在细胞连接处的分布增强,occludin/ZO-1、VE-cadherin/β-catenin之间的相互作用均较对照组显著增强,且以上Ang1处理所引起的SCMEC功能改变均可被wortmannin所拮抗。表明Ang1通过Akt活性调节SCMEC连接蛋白的分布及相互作用,从而影响体外BSCB的屏障功能。

To investigate the mechanism through which Ang1 regulates the barrier function of cultured SCMEC. Rat SCMECs and glial cells were isolated and the in vitro BSCB model was established by the bicameral culturing system. Cells were treated with Ang1 or Ang1＋wortmannin（the Akt inhibitor） and the protein level of p Akt, Akt and junction proteins were detected by Western-blot. Autoradiography was applied to detect the change of Akt activity. TEER in different groups was quantified to reflect the barrier function of SCMEC. The distribution of junction protein at cell contacts was detected by immunofluorescence. Co-immunoprecipitation was used to detect junction protein interactions. The results showed that both the Akt activity and TEER of the SCMEC increased significantly after Ang1 treatment. The distribution of ZO-1 and VEcadhirin at cell junctions and their respective interactions with occludin and β-catenin were also enhanced in Ang1 group. Furthermore, all the above effects of Ang1 were found to be antagonized by wortmannin. These results indicate that Ang1 could regulate the barrier function of BSCB in vitro through Akt activity.