体外原代培养胎鼠大脑皮层神经元NMDAR1亚基表达的发育性变化

目的 探讨体外原代培养胎鼠大脑皮层神经元NMDAR1（NR1）亚基表达的发育性变化。 方法 利用体外原代培养Wistar孕14～15d胎鼠大脑皮层神经元，采用免疫荧光法鉴定神经元的纯度和NR1亚基在神经元上的定位，采用流式细胞术和免疫印迹法（Western  blot）检测不同培养时间神经元NR1亚基的表达情况 结果 体外原代培养的胎鼠大脑皮层神经元纯度均达到90%以上，免疫荧光标记显示NR1亚基主要分布在神经元胞膜及树突干膜上，流式细胞仪检测培养1、2、3、4、6、9、12d神经元NR1亚基平均荧光强度分别为4.57±0.99、8.18±1.22、13.67±1.99、20.33±1.03、26.30±2.88、31.71±2.47、28.63±1.40，Western blot显示细胞膜蛋白中NR1亚基在1～2d表达微弱，3～6d表达逐渐增高,6d以后保持稳定。  结论 体外原代培养的胎鼠大脑皮层神经元NR1亚基有发育性变化，这种变化与神经元的成熟度有关。

Developmental expression of NMDAR1 subunit on cultured cerebral cortical neurons of fetal rats in vitro

Objective To investigate developmental expression of NMDAR1（NR1） subunit on cultured cerebral cortical neurons of fetal rats in vitro.  Methods  Cerebral cortical neurons were primarily cultured from pregnant 14～15d fetal rats.Purity of neurons and location of NR1 subunit were identified by immunofluorescence, and expression of NR1 subunit was determined by flow cytometry and Western blot.  Results The purity of neurons was more than 90%. Immunofluorescence showed that NR1 subunit is mainly located on the membrane of neuronic bodies and dendritic stems. Flow cytometry showed that average fluorescence intensity of NR1 subunit on 1d, 2d, 3d, 4d, 6d, 9d, and 12d was 4.57±0.99, 8.18±1.22, 13.67±1.99, 20.33±1.03, 26.30±2.88, 31.71±2.47, and 28.63±1.40,respectively. Western blot showed that the membrane protein NR1 subunit expression was weak on 1-2d, and gradually strengthened on 3-6d, then remained stably.  Conclusion Expression of NR1 subunit on cultured cerebral cortical neurons of fetal rats has developmental changes, which is related to neuronic maturity.