骨髓间充质干细胞移植入脑缺血大鼠侧脑室后的迁移及分化:免疫荧光标记

Migration and differentiation of bone marrow mesenchymal stem cells after transplantation into the lateral cerebral ventricle of ischemic rats

目的:骨髓间充质干细胞可以分化成神经细胞替代受损组织,并可分泌生长因子和营养因子来促进其体内细胞的存活和分化。实验将体外扩增和Hoechst33342标记的骨髓间充质干细胞移植入缺血性大鼠侧脑室内,观察干细胞在大鼠脑内的生存、迁移、分化情况及对神经功能恢复的影响。方法:实验于2004-12/2007-06在南京大学医学院附属鼓楼医院科研部完成。①取体质量120～160g的SD大鼠用于制备移植细胞;另取80只体质量250～300g的SD大鼠用于制作大脑中动脉闭塞模型。实验方法符合动物伦理学要求。②应用直接贴壁法分离纯化及扩增大鼠骨髓间充质干细胞,荧光染料Hoechst33342标记。③将造模成功的75只大脑中动脉闭塞大鼠按随机数字表法分为3组:模型对照组15只;磷酸缓冲液组30只;骨髓间充质干细胞移植组30只,将骨髓间充质干细胞立体定向移植到大鼠缺血侧脑室中。④术后1,3,6,12,24d测定大鼠神经功能损害评分,荧光显微镜下观察Hoechst33342标记的骨髓间充质干细胞在脑内的生存和迁移情况,采用免疫荧光法检测胶质原纤维酸性蛋白和微管相关蛋白2的表达。结果:①细胞移植后6d,12d骨髓间充质干细胞移植组大鼠的神经功能评分均显著低于磷酸缓冲液组(P<0.05)。②移植的骨髓间充质干细胞可在大鼠脑组织中存活,并向缺血区域迁移。③移植第24天Hoechst33342/微管相关蛋白2、Hoechst33342/胶质原纤维酸性蛋白双阳性细胞占Hoechst33342阳性细胞的百分率为(10.45±1.35)%,(8.73±1.38)%。结论:骨髓间充质干细胞可以在动脉闭塞局灶性脑缺血模型大鼠脑中存活,并向缺血区域附近迁移,在一定条件下可分化为神经元和星形胶质细胞,同时能促进局灶性脑缺血大鼠的神经功能恢复。

AIM： Bone marrow mesenchymal stem cells （BMSCs） could differentiate into nerve calls to substitute injured tissue, and could secrete growth factor and nutrition factor to promote the survival and differentiation of calls. In this study, the amplified and Hoechst33342-1abeled BMSCs in vitro were transplanted into lateral cerebral ventricle in ischemic rat to osbserve the survival time, migration and neural differentiation of BMSCs and the effect on neurological functional recovery. METHODS： The experiment was conducted in the Scientific Research Department of Drum Tower Hospital, Nanjing University Medical College from December 2004 to June 2007. ①SD rats of 120-160 g were selected for transplanted call preparation. Another 80 SD rats of 250-300 g were selected to establish models of middle cerebral artery occlusion. The experimental methods were accorded with the animal ethics. ②BMSCs were isolated, purified and amplified by direct attachment method, and labeled with Hoechst 33342. ③Successfully modeled 75 rats were randomly divided into model control group （n =15）, phosphate buffer group （n =30）, and BMSCs transplantation group （n =30）, in which Hoechst 33342-labeled BMSCs were transplanted into the lateral cerebral ventricle of rats. ④Neurological function injury severity was evaluated on days 1, 3, 6, 12 and 24 after transplantation. The survival and migration of Hoechst 33342-labeled MSCs were observed under fluorescent microcopy. The expressions of microtubule-associated protein-2 （MAP-2） and glial fibrilliary acidic protein （GFAP） of were detected using immunofluorescence staining. RESULTS： ①Compared to phosphate buffer group, the neurological function scores in transplantation group were significantly lower on the 6^th day after transplantation （P 〈 0.05）. ②The transplanted BMSCs survived in rat brain and migrated into ischemic area. ③About （10.45±1.35）% and （8.73±1.38）% Hoechst33342-1abled MSCs expressed MAP-2 and GFAP, respectively. CONCLUSION： BMSCs can survive and migrate into ischemic area of rats with middle cerebral artery occlusion. Part of MSCs can differentiate into astrocytes or neurons under certain conditions. MSCs could significantly improve the neurological recovery after MCAO in rats.