绿色荧光蛋白标记的骨髓间充质干细胞在脑缺血大鼠体内迁移的实验研究

Migration of enhanced green fluorescent protein labeled bone marrow after transplantation into rat cerebral infarct

目的:探讨基质细胞衍生因子-1α(SDF-1α)对骨髓间充质干细胞(BMSCs)在SD大鼠脑缺血后迁移至脑损伤区的影响。方法:构建大脑中动脉阻塞的脑缺血模型;50只大鼠随机分为脑缺血PBS对照组、脑缺血MSCs治疗组。将腺病毒携带的增强型绿色荧光蛋白(EGFP)标记的MSCs,在脑缺血1d后经尾静脉注射入大鼠体内;用实时定量PCR检测缺血半暗带区基质细胞衍生因子-1α(SDF-1α)的分泌;用流式细胞仪检测骨髓间充质干细胞上CXCR4的表达率;用荧光共聚焦显微镜扫描显示骨髓间充质干细胞的迁移。结果:GFP转染率大约87%转染骨髓间充质干细胞;实时定量PCR显示大鼠海马分泌的SDF-1α在1d时达到峰值,1-14d一直维持在较高的水平,14d后开始缓慢下降,而皮质分泌的SDF-1α在第3d开始缓慢上升,14d才达到峰值;流式细胞仪检测BMSCs表面的CXCR4有14%;GFP标记的BMSCs移植后6h发现聚集在大脑中动脉起始处(嗅球),在第3d后在丘脑等缺血半暗带区,在14d后皮质处的GFP+BMSCs已有明显的增加。结论:损伤组织SDF-1α浓度的升高与骨髓间充质干细胞迁移的增加可能存在一定的关系。

AIM ： To investigate the role of SDF - 1α in migrating of bone marrow stromal cells to the injured areas. METHODS： Ischemic brain lesion model was created in rats by permanent middle cerebral artery occlusion （MCAO）. 48 SD rats were divided randomly into 2 groups. Group 1 ： phosphate buffered saline （PBS 1 mL） for control （ n = 25） ; Group 2 ： BMSCs （2 × 10^6 ） were injected intravenously at 24 h after MCAO （ n = 24）. After propagated in BMSCs, AdS/F35 GFP （ green fluorescent protein） was infected to BMSCs. The expression of SDF - 1 oL （ stromal cell - derived factor - 1α） mRNA in the penrumbral tissue was assayed by real - time quantitative PCR. The expression of CXCR4 on MSCs was detected by flow cytometry. Confocal microscopy was used to detect the GFP - labeled MSCs migration. RESULTS：AdS/F35 GFP signals was observed in almost infected BMSCs. The expressions of SDF -1α mRNA in the thalamus and hippocampus of the ischemic brains were peaked at 3rd day after stroke, followed by a decrease at 14th day post - ischemia. The expression of SDF - let mRNA in the cortex of the ischemic brains was peaked at 7th day post - ischemia, still at high level at 14th day post - ischemia： The median percentage of surface CXCR4 expression in BMSCs was 14%. GFP labeled BMSCs were detected in the origination of the middle cerebral artery （olfactory area） at 6 h, after 3 days in the prenumbra tissue such as thalamus, and in the cortex more labeled cells were found after 14 d post - ischemia. CONCLUSION ： BMSCs can pass through the blood brain barrier of ischemic rats. Its mechanism might be associated with the expression of SDF - 1α in the ischemic brain.