摘要
目的探讨阿糖胞苷(Ara-C)在原代大鼠海马神经元早期纯化培养中的优化方法。方法新生1d SD大鼠海马神经元原代培养48 h后,不同浓度(0、2.5、5.0、7.5、10 mmol/L)Ara-C处理0、6、12、24 h,光镜下观察细胞形态,采用MTT检测细胞活性,并通过细胞形态学分析神经元纯度,筛选出最优纯化浓度与时间;采用免疫荧光及Western blot技术检测微管相关蛋白2(MAP2)进一步检测神经元纯度,台盼蓝染色分析神经元存活率。结果大鼠原代海马神经元培养48 h,5 mmol/L Ara-C处理24 h得到的神经元纯度及活性最佳;MAP2免疫荧光染色结果显示,Ara-C处理组神经元纯度为(74.28±10.13)%,显著高于对照组(34.82±8.15)%(P=0.000);Western Blot结果显示Ara-C处理组MAP2蛋白水平显著高于对照组(P=0.008);台盼蓝染色显示Ara-C处理组与对照组间海马神经元存活率无差异,分别为(93.2±3.82)%和(95.6±2.37)%(P=0.109)。结论在大鼠原代海马神经元的早期培养过程中,5 mmol/L Ara-C处理24 h得到的海马神经元的纯度与活性最佳。
Objective To investigate the optimum condition of Ara-C treatment in cell purifica-tionculture of rat primary hippocampal neurons. Methods Primary neurons in hippocampal region of new born （1 day after birth） SD rats were cultured for 48 h and then were treated with different doses （0 mol/L, 2.5 mol/L, 5.0 mol/L, 7.5 mol/L and 10 mol/L） of Ara-C for 0 h, 6 h, 12 h and 24 h. Cellular morphology was observed by microscope. Cell viabilities were determined by MTT assay and neuron purities were calculated by morphology. To further assess the neuron purities, MAP2 antibody were used by immunofluorescence and western blot and cell viability is measured by Trypan blue staining. Results Hippocampal neuron purity and cell viability was optimal when 5 mol/L Ara-C was added for 24 h. Neuron purity with Ara-C was better than control （74.28 ± 10.13）%VS （34.82 ± 8.15）%, （P=0.000））. MAP2 protein level in cells treated with Ara-C was higher than that in control cells （P=0.008）. The cell viabilities in both conditions were similar （（93.2 ± 3.82）%for Ara-C treatment VS （95.6±2.37）%for control treatment, （P=0.109））. Conclusion The optimum condition of Ara-C treatment in terms of neuron purity and cell viability was 5 mol/L for 24 h in cell culture of rat primary hippocampal neurons.
出处
《中国临床解剖学杂志》
CSCD
北大核心
2015年第6期681-684,689,共5页
Chinese Journal of Clinical Anatomy
基金
国家自然科学基金(81572222
30900725
81170682)
广东省自然科学基金(2014A030313333)
