新生大鼠脊髓源性神经干细胞的分离培养及分化鉴定

Sepration,cultivation and differentiation of neural stem cells from newborn rat spinal cords

目的:探讨分离、培养新生大鼠脊髓源性神经干细胞的方法并对其进行分化鉴定,为临床应用脊髓神经干细胞进行周围神经系统疾病的替代治疗奠定细胞学基础。方法:首次采用注射器灌注法分离新生24hWistar大鼠脊髓源性神经干细胞,神经细胞培养液调节细胞终浓度并进行培养,形成克隆后,传代。将传至第1代培养细胞加入含10%胎牛血清的DMEM/F12培养基诱导其分化。形态学方法观察细胞的生长状态,利用鼠抗Nestin抗体、鼠抗GFAP抗体、鼠抗MAP-2抗体进行分化的神经干细胞免疫组织化学鉴定。结果:新生大鼠脊髓源性神经干细胞可在体外培养条件下贴壁生长,在血清的作用下可分化为具有典型神经细胞形态、表达特异性组织蛋白Nestin、GFAP、MAP-2的神经细胞。结论:大鼠脊髓内可分离出脊髓源性神经干细胞,呈贴壁增殖状态,并有分化能力。

Objective To study the method of isolation and cultivation of neural stem cells from newborn rat spinal cords and identification of its differentiation, to establish cytological foundation for vicariousness therapy of peripheral nerveous system diseases by using spinal stem cells. Methods The neural stem cells were isolated by injector perfusion method from spinal cords of neonatal Wistar rat within 24 h, and induced by serum, the morphological method was used to observe the growth status of cells, anti-Nestin antibody, anti-GFAP antibody and anti-MAP-2 antibody were used for immunoeytochemistry test to identify cell differentiation. Results The status of neural stem cells from newborn rat spinal cords was adherence culture in vitro, and the cells could be induced by serum to diffentiate into neural cells with typical morphology and expressing tissue-specific protein Nestin, GFAP, MAP-2. Conclusion The neural stem cells can be isolated from newborn rat spinal cords with adherence proliferation status and they have potential of differentiation.