经静脉移植骨髓间质干细胞在脑缺血前后大鼠脏器组织中的分布

Distribution of intravenously grafted bone marrow mesenchymal stem cells in the viscera tissues of rats before and after cerebral ischemia

目的:骨髓章质干细胞通过局部直接注射或经静脉、动脉移植均可有效趋化到达脑缺血、损伤组织,但目前除了对到达靶部位的干细胞分布有一定了解外,对干细胞在非损伤组织或损伤组织之外的其它部位迁移分布与生存情况的研究较少。观察经静脉移植的大鼠骨髓间质干细胞在脑缺血前后大鼠体内的生存与分布,为干细胞移植研究提供实验基础和可能的理论依据。方法:实验于2006-05/2007-05在福建省神经病学研究所完成。实验材料:选取清洁级健康雄性近交系F344大鼠48只,体质量250～300g,由中国利学院上海实验动物中心提供,按随机数字表法分为3组;正常移植组、脑缺血前移植组、脑缺血后移植组,每组16只。实验过程中对动物的处置符合动物伦理学标准。实验方法:①体外培养扩增2月龄F344大鼠骨髓间质干细胞,传至第三四代备用。用带增强型绿荧光蛋白基因的慢病毒感染大鼠骨髓间质干细胞。②建立大鼠短暂大脑中动脉闭塞脑局灶缺血模型。取增强型绿荧光蛋白标记的大鼠骨髓间质干细胞,经股静脉移植入大鼠体内。正常移植组:细胞移植前大鼠未经任何处理;脑缺血前移植组:先在正常大鼠静脉移植细胞,24h后行大脑中动脉闭搴术;脑缺血后移植组:大鼠先行大脑中动脉闭塞术,24h后移植细胞。③实验评估:采用改良的神经功能缺损量表测定各组大鼠神经功能情况;取大鼠骨髓制作涂片,同时取脑、心、肺、肝、脾、肾、骨骼肌、小肠组织标本制作冰冻切片,在荧光显微镜下观察增强型绿荧光蛋白阳性细胞在各时间点的分布情况。结果:48只大鼠均进入结果分析。①脑缺血前移植组和脑缺血后移植组大鼠神经功能在移植后12周内均有明显恢复,但脑缺血后移植组恢复较快,术后1,3,12周明显优于脑缺血前移植组(P<0.01,P<0.05)。②在正常移植组和脑缺血前前移植组大鼠肺、骨髓、脾、小肠发现增强型绿荧光蛋白阳性细胞,而在其他组织未发现。在脑缺血后移植组大鼠脑半球缺血区发现大量增强型绿荧光蛋白阳性细胞聚集,在肺中也可见阳性细胞分布,在脑缺血对侧半球及其他组织中未发现。结论:移植前的脑损伤对移植的大鼠骨髓间质干细胞向脑局灶缺血损伤区迁侈,进而促进神经功能修复非常重要,否则,移植于细胞将在骨髓和脾脏较长时间停留且不易再次迁移。

AIM： The bone marrow mesenchymal stem cells administered intravenously,intra-arterially,or intracerebrally could preferentially migrate to the injured region of the brain.But little is known about the ability of bone marrow mesenchymal stem cells to migrate to the non-injured region.Our study was to observe the distribution and survival of intravenously grafted rat bone marrow mesenchymal stem cells in vivo before and after cerebral ischemia in rats,and provide the experimental evidence and possible theoretic explanation for study on stem cell transplantation. METHODS：Experiments were conducted in the Neurology Institute of Fujian Province from May 2006 to May 2007. Experimental materials： F344 rats were provided by Shanghai Laboratory Animal Center,Chinese Academy of Sciences. Totally 48 healthy adult male F344 rats weighing 250-300 g were randomly divided into 3 groups, group A,group B,and group C,with 16 rats in each.All animal procedures were performed in accordance with the guidelines provided by Laboratory Animal Committee. Experimental methods： ①The bone marrow mesenchymal stem cells were obtained from the male F344 rats of two months old ,cultured in vitro,and the third and fourth generation bone marrow mesenchymal stem cells were adopted.Ex vivo-expanded bone marrow mesenchymal stem cells were transduced with the lentiviral vectors which already carried enhanced green fluorescent protein gene. ②The rats were subjected to transient middle cerebral artery occlusion,then the behavior changes of rats were observed.The bone marrow mesenchymal stem cells marked with enhanced green fluorescent protein were infused into the femoral vein of rats. Bone marrow mesenchymal stem cell transplantation was carried out in rats of the group A. Bone marrow mesenchymal stem cell transplantation was carried out 24 hours before middle cerebral artery occlusion in the group B. Bone marrow mesenchymal stem cell transplantation was carried out 24 hours after middle cerebral artery occlusion in the group C. ③Neurological function was assessed by modified Neurological Severity Score. The tissue samples of brain,heart,lung,liver,spleen,kidney, skeletal muscle,small intestine and bone marrow of rats were collected,then the frozen sections and bone marrow smear were prepared.The distribution of enhanced green fluorescent protein positive cells was observed under the fluorescent microscope. RESULTS ：All the 48 rats were involved in the result analysis. ①Within 12 weeks after transplantation,the neurological function deficit in the group B and group C was obviously recovered,but the recovery was rapid in the group C. The recovery was significantly improved as compared with the group B in the first,third and twelfth week（P 〈 0.01, P 〈 0.05）. ②In the group A and group B,enhanced green fluorescent protein positive cells were found in bone marrow sample, lung,spleen and intestine samples,but not in the other tissues. In the group C,enhanced green fluorescent protein positive cells were localized to the ischemic region. Few marked cells were observed in the contralateral cerebral hemisphere or other tissues except a few in lung. CONCLUSION ：The cerebral injury before transplantation is very important and necessary for intravenously grafted Bone marrow mesenchymal stem cells migrating to ischemic focus,otherwise,the grafted bone marrow mesenchymal stem cells will settle down in bone marrow,spleen ,and never migrate to other tissues again.