骨髓神经组织定向干细胞的分离培养鉴定

Isolation,culture and identification of neural tissue-committed stem cells from bone marrow

目的:为临床中枢神经系统疾患的细胞移植及组织工程修复周围神经提供一种应用广泛、可来源于自体、无免疫排斥和伦理道德等问题的移植用种子细胞。方法:采用DMEM/F12(1∶1)无血清神经干细胞培养液,通过先贴壁、后悬浮的方法从大鼠骨髓中分离、培养单克隆生长的神经组织定向干细胞(neural tissue-committed stem cells,NTC-SCs),通过免疫细胞化学检测NTCSCs细胞球CXCR4和Nestin蛋白,以及细胞球自然分化后神经元β-TublinⅢ、MAP2ab以及神经胶质细胞CNPase、GFAP等蛋白的表达。结果:NTCSCs细胞球表达神经干细胞标志蛋白Nestin和组织定向干细胞标志蛋白CXCR4;NTCSCs细胞球在含15%胎牛血清的DMEM培养液中可自然分化,免疫荧光显示神经元和神经胶质蛋白β-TublinⅢ、MAP2ab、CNPase、GFAP等标志物阳性。结论:本研究提供了一种操作简便、成本低廉的NTCSCs的分离培养方法,NTCSCs作为种子细胞为脑、脊髓等神经系统疾患和创伤的修复和治疗提供了广阔的应用前景。

Objective： To provide autologous transplantation cells without immune rejection and ethical issue for neural grafting in the treatment of neurological diseases. Methods： Neural tissue-committed stem cells （NTCSCs） were isolated and expanded by suspensive growth from adherent cells in bone marrow in DMEM/F12 serum-free medium. NTCSCs were identified by immunocytochemistry for the markers of neural stem cells, and neural lineages after differentiation of cellular sphere. Results： The NTCSC spheres expressed nestin, a neural stem cell marker, as well as CXCR4, a marker for tissue-committed stem cells. The NTCSC spheres were naturally differentiated in DMEM with 15% fetal bovine serum. The differentiated cells expressed β-tublinⅢ, MAP2ab, CNPase and GFAP, markers of neural lineages. Conclusion： The NTCSC can be isolated from bone marrow and expanded in vitro, and it has a potential for treating neurological diseases of brain and spinal cord as a seeding cell.