胰酶快速消化大鼠皮层神经细胞原代培养方法分析

Short digestive time with trypsin in primary culture of rat cortical neuron

目的探索更简易可行的原代神经细胞培养方法。方法在文献报道方法的基础上,注射器抽吸制备神经细胞匀浆,0.125%胰酶(含0.2‰EDTA)短时消化脑组织3～5min,加入含10%胎牛血清的DMEM培养液,接种于经多聚赖氨酸(poly-L-lysine,PLL)预处理的六孔板上,24h后加入终浓度为10μM的阿糖胞苷(arabinofuranoside cytosine,Ara-C)抑制胶质细胞生长,48h后更换培养液终止Ara-C的作用。以后每2～3d更换培养液,神经细胞在含95%CO2培养箱中培养7d后,用神经元特异烯醇化酶(neuron-specific enolase,NSE)染色鉴定神经元纯度。结果培养细胞成熟后,神经细胞生长良好,NSE染色鉴定证实,神经元纯度超过95%。结论改良后的胰酶快速消化原代神经细胞培养方法,可减少实验动物用量,减少细胞损伤及污染机会,方法确切可行。

Objective To explore a more convenient and reliable method of primary cortex neuron culture. Methods On the basis of previous methods, syringe pumping cortex in and out was used to make neuron homogenate. Secondly, 0. 125% trypsin with 0.2%0 EDTA was add into the neuron homogenate to digest the connective tissue for 3- 5 minutes. Neuron ho- mogenate was seeding onto poly-D-lysine coated six-well plates with DMEM and 10% fetal bovine serum. Twenty-four hours after seeding, the medium was replaced and 10 μm arabinofuranoside cytosine （Ara-C） was added into the cultured medium. Forty-eight hours after seeding, the cultured medium changed again to stop the effect of Ara-C. Lastly, the cultured medium was changed every 2-3 days until neuron culture was incubated with 95% CO2 for 7 days into confluence. Neuron-specific eno- lase （NSE） staining was used to identify the purity of cultured neurons. Results More than 95% of neuron purity was detected by NSE staining. Conclusion The improved primary neuron culture method with low concentration of trypsin and short diges- tive time can reduce the quantity of animal used, cell lesions and pollution chance. It is a convenient and reliable method to get primary neuron culture in vitro.