摘要
背景:近年研究发现移植的骨髓间充质干细胞能向颅内创伤、脑卒中、炎症和变性疾病等病灶部位迁移,进而发挥治疗作用,但对于其向病灶定向迁移的具体机制还不十分清楚。目的:探讨基质细胞衍生因子1及其受体CXCR4在移植的骨髓间充质干细胞趋向缺血脑组织迁移中的作用。设计、时间及地点:细胞学体内实验,于2008-02/2009-02在解放军第三军医大学新桥医院中心实验室进行。材料:骨髓标本取自解放军第三军医大学附属新桥医院血液科收治的15～40岁正常或原发病未累及骨髓患者,三四月龄健康雄性SD大鼠72只由解放军第三军医大学野战外科研究所实验动物中心提供。方法:密度梯度离心贴壁筛选法分离纯化、体外培养人骨髓间充质干细胞。54只大鼠参照Nagasawa线栓法制备局灶性脑缺血再灌注模型,剩余18只作为假手术组,仅插入线栓10mm。模型组及假手术组大鼠各取9只,分别于造模后第2,4,8天,采用Real-timePCR和免疫组织化学法定量分析缺血脑组织基质细胞衍生因子1表达变化。剩余36只脑缺血再灌注模型鼠随机分为细胞移植组、溶液对照组,18只/组,于再灌注后24h分别从尾静脉缓慢注入1mL人骨髓间充质干细胞悬液(含2×109L-1个细胞)或1mLPBS。主要观察指标:人骨髓间充质干细胞CXCR4 mRNA和蛋白的表达,缺血再灌注后脑组织基质细胞衍生因子1mRNA和蛋白表达变化,免疫组织化学检测人骨髓间充质干细胞向缺血脑组织的迁移和分布。结果:RT-PCR结果发现人骨髓间充质干细胞表达CXCR4 mRNA,免疫细胞化学染色发现CXCR4主要表达于人骨髓间充质干细胞的胞膜和胞浆。脑缺血再灌注损伤后2,4,8d,趋化因子基质细胞衍生因子1mRNA水平呈上升趋势,与假手术组比较差异有显著性意义(P<0.05)。经静脉移植的人骨髓间充质干细胞定向迁移到脑损伤区域,并大量分布于基质细胞衍生因子1高表达的缺血半暗区,损伤侧大脑半球人骨髓间充质干细胞数量显著高于对侧半球(P<0.01)。结论:基质细胞衍生因子1及其受体CXCR4参与并促进人骨髓间充质干细胞向脑缺血再灌注损伤区的迁移。
BACKGROUND： Recent research has shown that transplanted bone marrow mesenchymal stem cells （BMSCs） migrate to the injured regions and exert their therapeutic effects in cases of intracranial trauma, stroke, inflammation and degenerative disease. The specific mechanisms involved in their migration to lesions are still to be fully elucidated. OBJECTIVE： To explore the effects of stromal cells derived factor-1 （SDF-1） and its receptor CXCR4 on the migration of transplanted BMSCs to ischemic brain lesions. DESIGN, TIME AND SETTING： The cytological in vivo study was perfonned at the Central Laboratory, Xinqiao Hospital, Third Military Medical University of Chinese PLA from February 2008 to February 2009. MATERIALS： Bone marrow samples were obtained from normal or primary affection non-involved bone marrow patients aged 15 40 years at the Department of Hematology, Xinqiao Hospital, Third Military Medical University of Chinese PLA. A total of 72 healthy male Sprague Dawley rats aged 3-4 months were supplied by the Experimental Animal Center, Research Institute of Surgery, Third Military Medical University of Chinese PLA. METHODS： Human BMSCs were isolated by combination of gradient centrifugation and different adherent time method. The transient middle cerebral artery occlusion （MCAO） was induced using intraluminal vascular occlusion in 54 rats, based on the method described by Nagasawa et al. The remaining 18 rats served as sham operation group, only inserted with thread for 10 mm depth. At 2, 4 and 8 days after cerebral ischemia, the expression of SDF-1 in the ischemic brain was determined by real time RT-PCR and immunohistochemistry in 9 rats from either group. The remaining 36 rat models of cerebral ischemia/reperfusion were equally and randomly assigned into a cell transplantation and solution control groups. 1 mL human BMSCs （2×10^9/L cells） or 1 mL phosphate buffered saline were slowly infused through the caudal vein at 24 hours following reperfusion. MAIN OUTCOME MEASURES： CXCR4 mRNA and protein expression in human BMSCs was determined. SDF-1 mRNA and protein expression following ischemia/reperfusion were detected. Migration of transplanted human BMSCs into the damaged region was observed through immunohistochemistry. RESULTS： RT-PCR showed that human BMSCs were positive for CXCR4 mRNA. Immunocytochemistry revealed that CXCR4 mainly expressed in cell membrane and cytoplasm of human BMSCs. At 2, 4 and 8 days following cerebral ischemia/reperfusion, SDF-1 mRNA levels showed an increased tendency, and showed significant difference compared with the sham operation group （P 〈 0.05）. The transplanted hMSCs were mainly distributed in the ischemic penumbral area with high SDF-1 expression. Number of human BMSCs in the damaged hemisphere was significantly higher than in the other hemisphere （P 〈 0.01）. CONCLUSION： SDF-1/CXCR4 is involved in the directional migration of transplanted BMSCs to the ischemic damaged brain region.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第19期3719-3724,共6页
Journal of Clinical Rehabilitative Tissue Engineering Research
