阿糖胞苷在大鼠皮质神经元细胞培养中的适宜介入时间

发布时间：2018-04-04 07:00

  本文选题：阿糖胞苷　切入点：大脑皮质　出处：《中国组织工程研究》2017年12期

【摘要】：背景:由于中枢神经系统疾病研究需求,实验室常用有毒性的阿糖胞苷获得纯度较高的神经元细胞,然而阿糖胞苷的介入时间鲜见研究。目的:验证终浓度为10μmol/L阿糖胞苷在大鼠皮质神经元原代细胞培养中的适宜介入时间。方法:采用神经元专用培养基Neurobasal+B27进行大脑皮质组织原代神经元培养,分别于接种细胞后12,24,36,48 h加入终浓度为10μmol/L阿糖胞苷。每48 h半量换液。于7 d后倒置的显微镜下观察神经元形态;采用免疫细胞化学的方法对神经元特异性烯醇化酶进行免疫化学染色,鉴定神经元细胞的纯度和成熟度;计算机多功能图像分析系统对所有神经元特异性烯醇化酶阳性细胞进行形态学计量分析。结果与结论:(1)接种细胞后24 h加入阿糖胞苷,神经元纯度在90%以上,分化成熟神经元细胞占比89.00%,神经元细胞胞体面积最大,突触最长,胞质透亮,核大而明显,细胞体周围折光性强,立体感强,突起三四个,具有三四级分叉,形成良好的网络结构,非神经元细胞较少,神经元纯度及细胞状态最佳;(2)结果表明,阿糖胞苷介入培养过程越早,神经元细胞纯度越高,但阿糖胞苷对神经元细胞分化成熟的影响也会越明显;采用neurolbasal培养基,于接种细胞后24 h加入阿糖胞苷,可以获得理想纯度,成熟度较好,生长状态较好的大脑皮质神经元。
[Abstract]:Background: due to the need of central nervous system disease research, the toxic cytarabine is commonly used in laboratory to obtain high purity neuronal cells. However, the interventional time of cytarabine is rarely studied.Aim: to investigate the optimal interventional time of cytarabine (10 渭 mol/L) in primary culture of rat cortical neurons.Methods: primary neurons of cerebral cortex were cultured on Neurobasal B27 neuron culture medium. The final concentration of cytarabine was 10 渭 mol/L after inoculation.The liquid was changed every 48 hours.The morphology of neurons was observed under inverted microscope 7 days later and the purity and maturity of neuron cells were identified by immunocytochemical staining of neuron-specific enolase.The morphometric analysis of all neuron-specific enolase positive cells was performed by computer multifunctional image analysis system.Results and conclusion 24 hours after inoculation, cytarabine was added to the cells, the purity of neurons was over 90%, the proportion of differentiated neurons was 89.00, the area of neuronal cell body was the largest, the synapse was longest, the cytoplasm was clear, the nucleus was large and obvious.The results showed that there were three or four processes with three or four degrees of bifurcation, a good network structure, fewer non-neuron cells, and the best purity and state of the neurons.The earlier the cytarabine interventional culture process, the higher the purity of neuronal cells, but the more obvious the effect of cytarabine on the differentiation and maturation of neuronal cells. Cytarabine was added in neurolbasal medium 24 hours after inoculation.The cortical neurons with ideal purity, good maturity and good growth state can be obtained.
【作者单位】： 保定市第一中心医院神经外科;河北大学医学院;北京理工大学生命学院;解放军军事交通学院第九队;
【基金】：河北省自然科学基金(B2015201161) 国家自然科学基金(81502477)~~
【分类号】：R741
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本文编号：1708868