神经干细胞植入大鼠海马中的迁移

MIGRATION OF THE NEURAL STEM CELLS TRANSPLANTED INTO THE RAT HIPPOCAMPUS

目的 :观察成鼠神经干细胞移植入切割海马伞侧海马和正常侧海马后的存活和迁移情况。方法 :取成鼠前脑室下带 (SVZ)组织制成单细胞悬液 ,接种于含 EGF和 b FGF的 DMEM/ F12 (1∶ 1)无血清培养基中 ,待神经球形成后 ,用有限稀释法进行单细胞克隆。将单克隆细胞球进行消化、分离 ,加入 Brd U标记并扩增成大量来源于同一细胞的亚细胞系神经干细胞克隆球。取 SD大鼠 ,切割其右侧海马伞 ,术后 14天 ,将标记有 Brd U的神经干细胞植入两侧海马齿状回中。移植术后 1月 ,取脑冰冻切片 ,作 Nissl染色和 Brd U免疫荧光检测。结果 :Nissl染色片两侧海马齿状回中有许多大小不等的深染细胞沿颗粒下层排列。两侧移植区中的 Brd U免疫荧光标记细胞均从移植区沿海马颗粒下层迁移 ,形成一明显的迁移带。切割海马伞侧海马内的 Brd U免疫荧光亮点密度明显大于正常侧海马内的免疫荧光亮点密度。免疫荧光片 Nissl复染发现齿状回内的深染细胞与 Brd U免疫荧光亮点相对应 ,在切割海马伞侧海马齿状回内有大胞体的神经元样细胞 ,而正常侧较少见。结论 :移植至海马齿状回中的神经干细胞能存活 ,并可沿正常海马齿状回中固有的神经干细胞迁移路径即颗粒下层迁移。

Objective: To investigate the survival and migration of the neural stem cells (NSCs) transplanted into the bilateral hippocampi of adult rat with unilateral hippocampal fimbria transected. Methods: Single cell suspensions were prepared from the subventricular zone of adult rat brain and were plated into serum-free DMEM/F12(1∶1) medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). After formation of the primary neurospheres, single cell clone was obtained with limited dilution method. Then the single cell clone spheres were digested and dissociated, and 5-Bromo-2-deoxy-uridine(BrdU) was added into the culture medium to label the NSCs. Lastly, a number of neurospheres were proliferated. Meanwhile the right hippocampal fimbria of adult rat was transected. Two weeks later, NSCs labeled by BrdU were transplanted into the bilateral hippocampi of the right-hippocampal fimbria-transected rats. At a month after transplantation operation, Nissl staining and immunohistofluorescence technique were used to detect the BrdU-labeled cells in the hippocampi. Results: BrdU-labeled cells in the hippocampi migrated along the subgranular layer of dentate gyrus from graft area. The density of BrdU-labeled cells in the hippocampal fimbria-transected hippocampus was higher than that in the intact hippocampus. The BrdU positive cells in immunofluoresent slice were corresponding to the deep-dyed cells in Nissl slice. There were some neuron-like cells in each hippocampal fimbria-transected hippocampus but fewer in the intact side. Conclusion: The grafted NSCs can survive well and migrate along the subgranular layer of dentate gyrus that is the inherent route for NSCs migration in host brain hippocampus, and more grafted NSCs survived in the hippocampal fimbria-transected hippocampus than that in the intact hippocampus.The above results suggested that some substance expressed strongly for the survival, migration and differentiation of the grafted NSCs in the hippocampal fimbria-transected hippocampi.