胚胎海马来源干细胞移植治疗缺血性脑梗死的研究

目的： 对脑梗死大鼠进行胚胎海马来源的干细胞移植治疗，观察其能否在梗死灶部位分化为成熟的神经元而发挥神经替代作用，并最终改善动物的肢体功能。方法: 从绿色荧光蛋白(GFP)转基因SD胚胎大鼠的海马提取细胞进行体外培养，免疫荧光染色观察这些细胞的特征。光化学法制作大脑皮层局灶缺血梗死模型, 即光血栓皮层损伤(PCI)。PCI后1 d将20只SD大鼠随机分为干细胞移植组和对照组，前者在梗死灶附近植入胚胎海马来源的干细胞，而后者不进行干细胞移植。在PCI后1、7、14、21 d，用旋转实验对动物的肢体功能进行测试和评分。在PCI后3周和12周，以抗神经元核抗体(NeuN)染色成熟的神经元，观察移植干细胞的存活及其分化为各种亚型神经细胞的情况。结果: 来自SD大鼠胚胎海马的细胞明显表现出神经干细胞的特征。移植的细胞可以在脑梗死动物模型中至少存活12周，并能分化为成熟神经元、星形胶质细胞等亚型的神经细胞。与移植后3周相比，12周时的NeuN+/GFP+ 密度和移植物体积均有所减少(P<0.05)。旋转实验结果表明，在PCI后7、14、21 d，移植组动物在平衡木上的停留时间均显著长于对照组(P<0.01)。结论: 来源于胚胎海马的细胞具有神经干细胞特征，其被移植入脑梗死灶周围后能存活12周以上，并可以分化为成熟的神经细胞，这可能与动物运动功能的改善有关。该结果提示由移植物分化的神经细胞可能对受损宿主细胞发挥了替代作用。

Survival and neuronal differentiation of transplanted stem cells derived from embryonic hippocampus in adult rats with stroke lesions

AIM: To study whether exogenous neural stem cells (NSCs) derived from embryonic hippocampus differentiates into the neurons after transplanted into the infarct periphery of the brain in a stroke model and to further investigate the behavioral improvement in the rats.METHODS: The NSCs were prepared after isolated from the embryonic hippocampus of green fluorescent protein (GFP)-transgenic rats and cultured. The NSCs were identified using nestin and doublecortin(DCX) as markers. The cortical infarction in rats was induced using photochemical method, named photothrombotic cortical injury (PCI). Twenty adult rats were randomly divided into NSC transplantation group (NSC group) and control group. The cultured NSCs were transplanted into the infarct periphery of the rats in NSC group, and nothing in control group at first day was applied after PCI. The locomotor behavior of animals was checked using the rotarod test at 1st, 7th, 14th and 21st d after PCI. The survival and differentiation of transplanted NSCs were evaluated by immunocytochemical method at 12th week after PCI.RESULTS: The cells derived from the embryonic hippocampus significantly expressed the markers of NSCs. The grafted cells survived at least 12 weeks in the infarct periphery of adult rats and differentiated into mature glial cells and neurons. The density of NeuN+/GFP+ and volume of grafts at 12th week were less than those at 3rd week after transplantation (P<0.05). The time standing on the rod was longer in NSC group than that in control group at 7th, 14th and 21st d after PCI (P<0.01).CONCLUSION: The NSCs derived from embryonic hippocampus survive in the infarct periphery of adult rats up to 12 weeks and differentiate into mature neurons, which might be associated with the improvement of locomotor behavior of stroke animals. The neuronal replacement of neurons differentiated from NSCs may be the underlying mechanism.