大鼠原代皮质神经元无血清培养的影响因素及优化培养方法

Influencing Factors and Optimizing Cultivating Conditions of Primary Cortical Neurons of Rat in Serum-Free Culture

目的探讨大鼠原代皮质神经元无血清培养的影响因素及优化培养方法。方法采用原代神经元无血清培养,按胎鼠、24 h新生鼠、5 d新生鼠及不同取材环境温度(20℃、30℃)下胰蛋白酶消化0 min、5 min、15 min进行分组。应用锥虫蓝拒染法检测接种时神经元存活率,神经元特异性烯醇化酶(NSE)免疫组织细胞化学染色法检测各组所得神经元纯度,倒置显微镜动态观察各组神经元形态变化。结果胎鼠组和24 h新生鼠组原代神经元状态良好,2组细胞纯度[(91.30±1.03)%、(89.50±1.78)%]和存活率[(98.20±0.58)%、(97.10±0.98)%]比较差异均无统计学意义;5 d新生鼠组神经元纯度为(82.00±1.25)%,存活率为(92.87±1.56)%,均低于胎鼠和24 h新生鼠组(Pa<0.05)。缩短取材操作时间,结合环境温度适当调整消化时间可提高神经元细胞纯度和细胞活性:环境温度20℃,消化15 min,30℃消化5 min,细胞存活率最高,分别为(98.20±0.58)%、(96.70±0.64)%。结论 24 h新生鼠原代神经元无血清培养操作性强、省时,优化神经元培养过程影响因素(温度、消化时间等)可提高神经元细胞的纯度和存活率。

Objective To explore the influencing factors for the purity and viability of primary, cuhured cortical neurons of rat,and optimize the separating and cultivating conditions of the eortical neurons. Methods The primary cotical neurons were cultured in a serum - free culture system of B27 - supplemented neurubasal medium. The differences in purity and viability of primary cultured neurons between embryonic rat group and newborn rat [ postnatal 24 h and 5 d] group were evaluated by morphology,immunocytochemistry of neuron - specific enolase （NSE） and trypan blue staining. The changes of neurons purity and viability in difl＇erent trypsin digestion time（0 rain,5 min and 15 min） at different environment temperatures（20℃ and 30℃ ） were assessed by immunocytochemistry and trypan blue staining. Results The primary cultured neurons from fetal and newborn rats grew well. There was no significant diflhrence in embryonic rat and postnatal 1 d newborn rat group[ （91.30± 1.03）%, （89.50±1.78）% respectively in purity;and （98.20 -0.58）%, （97.10 ±0.98）% respectively in viability]. The neurons from 5 - days newborn rat were inferior to that from fetal and 1 - day newborn rat in purity and viability[ （82.00± 1.25） % and （92.87± 1.56）% respectively ]. Shortening operation time and adjusting digestion time according to the environment temperatures could improve neuronal viability：A digestion for 15rain at the environment temperature of 20℃ or for 5 min at 30% couht acquired cells with higher viability [ （98.20 ± 0.58 ） % and （96.70 ±0.64 ） % respectively ]. Conclusions It is an easy, practical choice to culture priamry cortical neurons from postnatal 1 d newborn rats. Optimizing the separating and cultivating condition （environment temperatures, digest time ,et al. ） will improve the neurons purity and viability.