中文摘要：

背景：目前临床上尚未发现完全治愈脊髓损伤,使其达到结构及功能恢复的治疗方法。已有的神经营养因子治疗方法促进神经细胞再生的效果有限,而干细胞移植治疗可能是现阶段修复脊髓损伤的有效方法之一。目的：无血清悬浮分离培养法培养胎鼠脊髓源性神经干细胞,采用形态学、免疫荧光技术和多向分化能力进行神经干细胞鉴定。方法：应用悬浮分离培养法,培养纯化孕13.5 d C57BL/6胎鼠的脊髓源性神经干细胞。倒置显微镜观察细胞形态学变化;CCK-8法检测细胞的增殖能力。免疫荧光技术检测Nestin和Sox2表达;使用自然分化法验证第4代脊髓源性神经干细胞多向分化特性,明确其分化能力。结果与结论：采用无血清悬浮分离培养法可以顺利获取脊髓源性神经干细胞。培养的脊髓源性神经干细胞增殖活性良好,高表达Nestin和Sox2,且两种标记物与DAPI核染高度均匀吻合,说明细胞纯度高。进一步诱导分化显示,细胞可以向GFAP和Tuj1阳性细胞分化,证明脊髓源性神经干细胞分化潜能较好。该实验可建立一套体外培养脊髓源性神经干细胞分离、培养、纯化及鉴定体系,为后续神经干细胞的应用研究奠定基础。

英文摘要：

BACKGROUND： Until now, there is yet no complete recovery from spinal cord injury in terms of structure and functional recoveries. Neurotrophic factors have limited effects on nerve regeneration. Currently, stem cell transplantation may be an effective way to repair spinal cord injury. OBJECTIVE： To separate, cultivate and purify mouse spinal cord-derived neural stem cells using serum-free suspension method followed by morphological observation, immunofluorescence technology and multi-lineage differentiation experiments. METHODS： By using the suspension culture method, mouse spinal cord-derived neural stem cells at embryonic day 13.5 were cultured and purified. Cell morphology changes were observed under inverted microscope. Cell proliferation ability was detected using cell counting kit-8. Nestin and Sox2 expression was detected by immunofluorescence technology. Multilineage differentiation of spinal cord-derived neural stem cells at passage 4 was detected by natural differentiation method in order to prove the differentiation ability. RESULTS AND CONCLUSION： Serum-free medium suspension culture method was successfully applied to separate spinal cord-derived neural stem cells. Cultured cells had good proliferative ability and highly expressed Nestin and Sox2 that was in accordance with the results of DAPI nucleus staining, suggesting the high purity of cells. After induction, the cells could express both Tuj1 and GFAP, indicating the cells had good differentiation potential. This experiment has successfully established the isolation, culture, identification system of spinal cord-derived neural stem cells, providing experimental basis for subsequent studies of neural stem cells.