新生大鼠脊髓运动神经元的纯化培养与鉴定

Spinal motor neurons from neonatal rats: purification, culture and identification

背景:神经元是有丝分裂后的细胞,体外培养很难存活。脊髓运动神经元的分离、纯化和培养同时也是细胞培养的技术难点。目的:建立新生大鼠脊髓运动神经元培养体系,并予以分类鉴定和测定其纯度。方法:取新生大鼠脊髓腹侧组织分离成细胞悬液,经密度梯度离心及差速贴壁后纯化培养,采用免疫细胞化学双标染色法对培养盖片上的细胞予以鉴定、分类,结合Hoechst荧光染核,计数各细胞成分的含量。结果与结论:细胞贴壁生长良好,神经元占85.8%,其中运动神经元达71.6%,星形胶质细胞占7.8%,NF200和胶质纤维酸性蛋白染色皆为阴性的细胞占6.4%。结果说明,取新生大鼠腹侧脊髓组织结合密度梯度离心及差速贴壁接种法可以培养出高纯度的脊髓运动神经元。

BACKGROUND：neurons are post-mitotic cels that are difficult to survive. Isolation, purification and culture of spinal motor neurons are technical difficulties of cel culture technology. OBJECTIVE：To establish the culture system of spinal motor neurons from neonatal rats and to identify and determine the purity of spinal cord neurons. METHODS： Ventral spinal cord tissues from neonatal rats were made into cel suspension that was subjected to density gradient centrifugation and differential adherence folowed by purification culture. Then, the cels on cover plates were identified and classified using immunocytochemical double staining method, and the content of cel components was calculated in combination with fluorescent Hoechst nuclear staining. RESULTS AND CONCLUSION：The cels adhered wel, and the neurons accounted for 85.8%, among which, motor neurons reached 71.6%, astrocytes accounted for 7.8%, cels negative for neurofilament 200 and glial fibrilary acidic protein were 6.4%. These findings indicate that the ventral spinal cord tissues from neonatal rats combined with density gradient centrifugation and differential adherence can develop high-purity spinal motor neurons.