骨髓基质细胞在大鼠纹状体出血模型脑内的存活及迁移.docVIP

骨髓基质细胞在大鼠纹状体出血模型脑内的存活及迁移 【摘要】 目的 观察骨髓基质细胞(MSCs)在大鼠纹状体出血模型脑内的存活及迁移特点，探讨其移植于出血脑内的可能性及适宜条件。方法 用密度梯度离心法分离培养成年SD大鼠的MSCs，流式细胞仪检测细胞表面抗原CD11b、CD45和CD90以确定其纯度，细胞大量扩增后用逆转录病毒载体转染增强型绿色荧光蛋白(EGFP)标记基因。胶原酶加肝素纹状体注射法制备大鼠脑出血模型，行为学检测成功后，将细胞悬液5μL立体定位植入脑出血模型大鼠出血灶周边。不同时间分批灌杀大鼠，取脑作冰冻切片，免疫组织化学法检测移植细胞的EGFP表达。结果 流式细胞仪检测发现CD90+/CD45-/CD11b-细胞达到75%。细胞在体外能够稳定地表达EGFP。MSCs在脑出血灶周边至少可存活4周，这些细胞形态多样，主要分布于出血灶周边，并向周围脑实质迁移，大多数细胞定居于血管周围。结论 分离培养的MSCs纯度高，增殖能力强，可高效表达EGFP，移植到脑出血灶周边可存活并向周围迁移。 【关键词】 脑出血 纹状体 骨髓基质细胞 移植 存活 大鼠 　　Abstract： Objective To explore the surviving and migration of Bone Marrow Stromal Cells(MSCs) in brain of the striatal hemorrhage model of rat, and to investigate the possibility and appropriate condition of transplanting MSCs into brain of the striatal hemorrhage model. Methods MSCs was collected from adult SpragueDawley(SD) rats by density gradient centrifugation method. Cell surface antigen CD11b, CD45 and CD90 were measured in order to estimate the purity of MSCs. After being expanded, transduction of MSCs with a retroviral vector containing the enhanced green fluorescent protein (EGFP) cDNA was performed. The striatal hemorrhage animal model was established by injecting collagenase and heparin into the striatum. After praxiology test succeeded, 5μl MSCs suspension was stereotactically transplanted into the brain of striatal hemorrhage model. Then rats were killed in batches at different time, and brain sections were prepared with a cryostat. Here, we examined the surviving and migration of MSCs in the area around hemorrhage focus by examining EGFP expression of transplanted cells by immunohistochemical methods. Results Cell surface antigen test showed that CD90+/CD45－/CD11b－ cells added up to 75%. MSCs expressed EGFP steadily in vitro, and survived for 4 weeks after being injected in the area around hemorrhage focus of the striatal hemorrhage model. In the hemorrhage focus, a few surviving MSCs had a multiple morphological appearance, migrated into the area around the hemorr