新生大鼠脊髓运动神经元的纯化培养与鉴定

发布时间：2019-03-08 12:29

【摘要】：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。
[Abstract]:Background: neurons are mitotic cells that are difficult to survive in vitro. The isolation, purification and culture of spinal motoneurons are also the technical difficulties in the culture of spinal cord motoneurons. Objective: to establish the culture system of spinal motoneurons in neonatal rats and to classify and determine its purity. Methods: cell suspension was isolated from ventral tissue of spinal cord of newborn rats and purified and cultured by density gradient centrifugation and differential adhesion. The cells on the culture cover were identified and classified by immunocytochemical double labeling method. Combined with Hoechst fluorescence staining, the contents of cell components were counted. Results & conclusion: the adherent cells grew well, with 85.8% of neurons, 71.6% of motoneurons, 7.8% of astrocytes, 6.4% of cells negative for NF200 and glial fibrillary acidic protein (GFAP) staining, and 71.6% for motor neurons, 7.8% for astrocytes and 6.4% for NF200 and GFAP negative cells. The results showed that the spinal cord motoneurons of high purity could be cultured by density gradient centrifugation and differential adherent inoculation in ventral spinal cord of newborn rats.
【作者单位】：大连医科大学/大连医科大学附属第一医院;
【基金】：辽宁省自然科学基金(2013023035) 大连市科技计划项目(2013E15SF171)~~
【分类号】：R329.2

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