目的:探讨基质细胞衍生因子-1(SDF-1)/CXCR4轴在外源性骨髓间充质干细胞(MSC)向哮喘模型小鼠肺组织迁移的作用.方法:无菌条件下取GFP转基因小鼠骨髓MSC,体外扩增,鉴定.采用Transwell培养系统,观察0、50、100、150和200 ng/ml SDF-1和CXCR4阻断剂AMD3100对MSC体外定向迁移的影响.取60只雌性BALB/c小鼠,随机分为6组(n=10):PBS非哮喘组、MSC非哮喘组、PBS哮喘组、MSC哮喘组、SDF-1处理哮喘组、AMD3100处理哮喘组.哮喘组采用哮喘致敏液0.2 ml(含100 μg卵白蛋白)致敏,并使用卵白蛋白雾化吸入激发哮喘.非哮喘组在致敏和激发时均予以PBS处理.MSC处理组于哮喘激发前移植外源性MSC.SDF-1处理哮喘组在MSC移植前气管内注入SDF-1,AMD3100处理哮喘组注入AMD3100预先孵育的MSC.PBS哮喘组注射等量的PBS液.采用Westernablot和RT-PCR方法检测肺组织中SDF-1的表达水平,通过荧光显微镜观察表达GFP的外源性MSC在哮喘小鼠肺组织中的分布情况,比较SDF-1和CXCR4阻断剂AMD3100干预对MSC向肺组织迁移的影响.结果:Transwell实验显示MSC的迁移水平与SDF-1(0~150军ng/ml)成浓度依赖性.与正常小鼠比较,哮喘小鼠肺组织SDF-1表达增强.与MSC非哮喘组比较,MSC哮喘组小鼠肺部有更多MSC聚集.在哮喘肺组织中增加外源性SDF-1能够促进MSC向肺组织迁移.通过AMD3100阻断MSC的CXCR4可以明显减少MSC向肺组织的迁移水平.结论:SDF-1/CXCR4轴参与了MSC迁移到哮喘小鼠肺组织的过程.
Objective: To investigate the involvement of stromal cell-derived factor-1 (SDF-1)/CXCR4 axis in the migration of exogenous mesenchymal stem ceils (MSCs) to lungs of asthmatic mice. Methods: MSCs were obtained from green fluorescence protein -transgenic donor mouse under sterile condition, and then they were amplified and identified. The effects of different concentration of SDF-1 (0,50,100,150,200 ng/ml) on MSC migration in vitro were observed by using Transwell system. Sixty female BALB/c mice were randomly divided into six groups: PBS-nonasthmatic group, MSC-nonasthmatic group, PBS-asthmatic group, MSC-asthmatic group, SDF-1-MSC group, and AMD3100-MSC group. Allergenic agent (0.2 ml, including 100 /lg ovalbumin) was injected intraper- itoneally on day 0, 7 and 14, and ovalbumin was administered via inhalation 15-21 days to reproduce asthmatic mouse model. PBS was used instead of ovalbumin in the two nonasthmatie groups. Exogenous MSCs (1 × 106 cells) were administered intravenously to 4 experimental groups, except PBS-nonasthmatic group and PBS asth- matic group. In SDF-1-MSC group, SDF-1 was given by intratracheal instillation 30 minutes before MSC trans- plantation. In AMD3100-MSC group, MSC was cultured with AMD3100 for 30 minutes before transplantation. PBS was used instead of MSCs in PBS asthmatic group. Lung tissue was harvested 24 hours after the last inhalation. Protein and mRNA expressions of SDF-1 in lung tissue were determined by Western blot or RT-PCR. The distribution of exogenous MSCs with or without pre-injection of SDF-1 or pretreatment of AMD3100 in lungs was observed by fluorescence microscope. The effects of SDF-1 and CXCR4 blocker AMD3100 on migration of MSCs in lung were also observed. Results: The experiment on Transwell system showed that MSCs migrated towards SDF-1-containing medium in a concentration-dependent manner. A higher expression of SDF-1 was observed in mice with asthma compared to that of non-asthmatic group. The number of MSCs localized in the lu