神经干细胞在体外诱导向胆碱能神经元定向分化后移植治疗脊髓损伤

Treatment of spinal cord injury by transplanting cholinergic neurons after inducing and directionally differentiating neural stem cells in vitro

目的 研究神经干细胞向胆碱能神经元定向分化后对脊髓横断损伤的修复作用。方法采用GFP转基因孕鼠（E12～14d）的海马分离、培养神经干细胞并诱导其向胆碱能神经元方向分化。SD成年大鼠以显微剪横断脊髓制成SCI模型。将SD大鼠18只分为3组：A组注入DMEM／F12培养液；B组注入神经干细胞的细胞液，C组注入在体外定向诱导为胆碱能神经元的细胞液；3组实验动物均于细胞移植后采用BBB评分法定期评估运动功能。于移植后第8周末，取出相应的脊髓阶段，行ChAT免疫组织化学染色，光镜观察。结果 从海马分离的细胞群具有自我更新能力，表达nestin，胎鼠骨骼肌提取液可以诱导这些细胞中的11．8％分化成为胆碱能神经元，与对照组差异明显。B组和C组所有大鼠BBB评分在移植细胞3周以后明显高于A组（P〈0．05），C组所有大鼠BBB评分在移植细胞4周以后明显高于B组（P〈0．05）。在移植第8周末，冰冻切片中可见ChAT染色阳性细胞。结论 神经干细胞可以在体外诱导向胆碱能神经元定向分化，定向分化后移植应用在脊髓横断损伤治疗中，可明显改善运动功能。

Objective To observe the effects of transplanting cholinergic neurons after inducing neural stem cells on treating spinal cord injury.Methods Adopt hippocampus isolation of transgenic fetal rat（12-14d） to culture neural stem cells and induce them to differentiate into cholinergic neurons, SCI models were made by tronsecting spinal cords of adult Sprague-Daweley rat with microscissors. 18 Sprague-Daweley rats were divided randomly into three groups. Group A was injected DMEM/F12. Group B was injected cell sap of neural stem cell. Group C was injected cell sap of cholinergic neurons. Locomotor functions of three groups in the experiment were detected termly by BBB score after cell transplantation, By the end of the eighth week after transplantation, the spinal cord tissues were extracted, by immunohistochemical stain of CHAT. Results Cell line isolated from the hippocampus had ability of proliferation and expressed nestin. Extract from fetal rat skeletalmuscle can induce 11.8% of the isolated cell line to differentiate into cholinergic neurons. This was different from the control group（ P 〈 0.01 ） significantly, BBB scores of all the adult rats in group B and C were obviously higher than that in group A after 3 weeks after transplantation（ P 〈 0.05）. BBB score in group C was obviously higher than that in group B after 4 weeks after transplantation （ P 〈 0.05）. By the end of the eighth week after transplantation, CHAT positive cells were observed in the frozen section. Conclusion NSCs can differentiate directionally into cholinergic neurons in vitro. Transplantation for treating spinal cord injury, can stimulate the recovery of motor functions.