神经干细胞移植修复大鼠脊髓半切伤的研究

目的：观察神经干细胞移植修复脊髓半切伤的疗效并探讨移植最佳时机。方法：制备大鼠T13-L1脊髓半切伤模型，取胎龄13、5d胎鼠大脑组织，体外分离、培养、诱导神经干细胞，并采用免疫细胞化学技术分别检测NSC（neural stemcell）特征性标志Nestin（巢蛋白）表达和用血清诱导分化为大量神经元NSE（neuron specific enolas）和神经胶质细胞GFAP（glial fibrillary acidic protein）表达。用明胶海绵吸附BrdU（5-溴脱氧尿嘧啶）标记好的神经干细胞悬液移植到脊髓半横断处，移植时间选择损伤后立即移植、损伤后第9、14d。观察移植后的大鼠行为的变化，通过CBS（combine behavioral score）评分和爬网格实验评价大鼠的运动功能恢复情况，并进行免疫组化鉴定，光学显微镜观察移植神经干细胞的存活和迁移。结果：在上述条件下培养及传代的细胞不断分裂增殖，形成悬浮生长的呈Nestin阳性的神经球；用血清诱导分化为大量表达NSE阳性的神经元和GFAP阳性的神经胶质细胞。与对照组相比，神经干细胞移植组明显修复了损伤结构，改善了下肢的功能，尤其是第9d移植组。结论：神经干细胞移植促进了脊髓损伤后神经结构和功能的恢复，是治疗脊髓半切伤一种有效方案，考虑综合因素移植干细胞的最佳时机应选损伤后9d左右。

The study on neural stem cell transplantation for repairing hemi-sected spinal cord injury in rats

Objective:To investigate the effects of neural stem cells(NSCs)transplantation on the recovery of structure and function in hemi-sected spinal cord injury in rats. Methods:The model of hemi-sected spinal cord injury was made at T13-L1 vertebra level. Neural stem cells of rat embryo (E-13.5) were cultured in vitro. Neurospheres and differentiated cells were identified by immuncytochemisty. After differentiating into neural progenitors, NSCs were incubated with Brdu and absorbed with gelfoam sponge, and then grafted into the injury site of spinal cord. The time of transplantation was as follows: at once, the ninth day and the fourteenth day after injury. Neural morphological repairing and function recovery were investigated quantitatively and the locomotor functions were detected by CBS score and grid test. Results:Cells cultured continuously proliferated and formed neurospheres which were Nestin positive. Differentiated cells showed the shapes of neuron and astrocyte, which were NSE and GFAP positive respectively. Transplantation group improved morphological repairing significantly comparing with the control, especially in the ninth day transplantation group. Conclusions:Transplantation of neural stem cells can promote the structural and functional recovery of hemi-sected spinal cord. It is suitable to choose optimal time around 9 days for transplantation after injury.