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| SD大鼠乳鼠皮层神经元细胞原代培养模型的建立及鉴定 |
| Primary Culture and Identification of Cortical Neurons from Neonatal SD Rats |
| 投稿时间：2021-03-03 修订日期：2021-03-03 |
| DOI： |
| 中文关键词: SD大鼠乳鼠 皮层神经元 原代培养 纯度 钙离子信号 |
| 英文关键词: Neonatal SD rats Cortical neurons Primary culture Purity Calcium ion signal |
| 基金项目:国家自然科学基金项目(面上项目，重点项目，重大项目）; 北京市科技计划项目 |
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| 中文摘要: |
| 目的：探讨SD大鼠乳鼠皮层神经元细胞原代培养方法，并鉴定其培养效果，以期建立一种生物学功能良好的体外细胞实验模型。方法：取出生24h的SD大鼠乳鼠，分离出大脑皮层，在胰酶消化之前先进行离心，然后将胰酶消化后多次离心得到的细胞悬液接种于L-多聚赖氨酸包被的培养皿和共聚焦皿中，以加B27的Neurobasal-A培养基进行神经元细胞的原代培养，倒置显微镜下观察培养细胞的生长状态；通过免疫荧光组化的方法采用神经元标记物MAP-2进行神经元纯度的鉴定；在导入Fluo4-AM的原代神经元细胞，观察电刺激后胞内钙离子信号的变化，以验证神经元细胞的生理状态。结果：采用此方法培养的神经元细胞紧密贴壁、分散均匀、状态良好，神经元细胞周围突起相互连接形成网络；经MAP-2免疫荧光组化技术鉴定神经元的纯度达到95%以上；胞内钙离子信号的变化提示所培养的神经元具有良好的生物学功能。结论：该方法能获得纯度较高并且生物学功能良好的原代培养的SD大鼠乳鼠皮层神经元细胞。 |
| 英文摘要: |
| Objective: To develop a better method for culturing cortical neurons from neonatal SD rats. Methods: The cerebral cortex of 24-hour-old SD rats were isolated and centrifuged before trypsin digestion. The cell suspension obtained after trypsin digestion was planted into L-poly lysine coated culture dish or confocal dish. The primary culture of neurons was carried out in a culture medium contained B27 and Neurobasal-A. The growth of cultured cells was checked under inverted microscope. Immunofluorescence histochemistry was used to identify the purity of neurons by using microtubule-associated protein 2 (MAP-2) as a marker of neuron. The changes of intracellular calcium signal after electrical stimulation was observed in Fluo4-AM loaded primary neurons to verify the physiological state of neurons. Results: The neurons cultured with this method were closely adherent, evenly dispersed and in good condition. The neurites of the neurons were interconnected to form a network. MAP-2 immunofluorescence histochemistry showed that the purity of neurons was more than 95%. The stimulation-induced intracellular calcium signal indicated that the cultured neurons had good biological function. Conclusions: This method can make primary cultured cortical neurons of SD rats with high purity and good biological function. |
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