MMP-9促进间充质干细胞穿越血脑屏障的作用研究

Research on MMP-9 to promote mesenchymal stem cells migration through the blood-brain barrier

目的 探讨基质金属蛋白酶-9（MMP-9）开放血脑屏障（BBB）,促进大鼠间充质干细胞（rMSCs）穿越BBB的作用.方法 构建体外BBB模型,运用Transwell细胞迁移率实验明确MMP-9对体外BBB模型的穿透效果.体内实验：4周龄SD大鼠随机分为假手术组、缺氧缺血性脑损伤（HIBD）组、MMP-9处理组及MMP-9抑制剂（TIMP-1）干预组;HE染色观察各实验组不同时间点（0.5、1、3、7、14d）的大脑皮质病理变化和检测其脑含水量,EB值法检测BBB通透性;同时采用Western Blot测定大鼠皮质中MMP-9蛋白表达和免疫组织化学法测定大脑皮质内5-溴-2-脱氧嘧啶（Brdu）标记rMSCs阳性细胞数量.结果 Transwell细胞迁移率实验显示,缺氧状态下各组穿过小室的rMSCs数量均明显高于常氧,差异有统计学意义（P＜0.01）;MMP-9处理组穿过小室的rMSCs数量明显高于阴性对照组和TIMP-1干预组,差异有统计学意义（P＜0.01）.体内实验结果表明：与HIBD组相比,MMP-9处理组脑含水量、BBB通透性增加程度均有所提高;TIMP-1干预组的相应指标均有所降低.大鼠皮质中MMP-9蛋白表达水平具有时间依赖性,MMP-9处理组1～3 d时含量达到最高,明显高于HIBD组和TIMP-1干预组,差异有统计学意义（P＜0.01,P＜0.05）,之后表达开始回落,但仍有稳定表达.MMP-9处理组穿越BBB的Brdu阳性细胞数比HIBD组多,差异有统计学意义（P＜0.01,P＜0.05）.结论 大鼠缺氧缺血后MMP-9表达升高,可介导开放BBB,促进rMSCs穿越BBB.

Objective To explore the role of MMP-9 in blood-brain barrier （BBB） disruption and to promote rMSCs migration.Methods To investigate the effect of MMP-9 on the permeability of BBB,an in vitro model of BBB was established and performed Transwell experiments.To observe the pathologic changes of the cerebral cortex,rat brains from Sham treated group,HIBD group,MMP-9 treated group and TIMP-1 （intervention） treated group at different time points （0.5,1,3,7,14 d） were prepared and stained with Hematoxylin-Eosin and quantified the brain water content.Permeability of BBB examined by EB values measurement.MMP-9 protein level of rat cortex was detected by Western Blot.The number of Brdu labeled rMSCs in the rat cerebral cortex of each group was quantified by Immunohistochemistry （IHC）.Results Transwell experiments results showed that migration of rMSCs increased remarkably in hypoxic condition compared to that of normal control （P＜0.01）.The number of rMSCs migrated in the MMP-9 treated group was much more than that of negative control group and TIMP-1 group （P＜0.01）.The results of pathology showed that compared to HIBD group,brain water content and the permeability of the BBB were increased in MMP-9 treated group but reduced in TIMP-1 treated one.The expression of MMP-9 protein in MMP-9 treated group was reached the peak at 3 d point,which was higher than that of HIBD group and TIMP-1 group （P＜0.05,P＜0.01）.The quantity of Brdu labeled rMSCs crossed BBB in MMP-9 treated group was extremely higher than that of HIBD group （P＜0.05,P＜0.01）.Conclusions The expression level of MMp-9 protein was improved after hypoxic-ischemic.MMP-9 could facilitate the migration of rMSCs through vitro BBB and control the opening of BBB,which indicate that MMP-9 may facilitate the migration of rMSCs through BBB into brain.