探讨阿糖胞苷在原代大鼠海马神经元培养中的纯化方法

李昂; 晏芳; 李振林; 刘忠英; 张琳; 董为人; 牛红心; 王海红

doi:10.13418/j.issn.1001-165x.2015.06.014

中国临床解剖学杂志 ›› 2015, Vol. 33 ›› Issue (6) : 681-684. DOI: 10.13418/j.issn.1001-165x.2015.06.014

实验研究

探讨阿糖胞苷在原代大鼠海马神经元培养中的纯化方法

李昂¹，　晏芳¹，　李振林¹，　刘忠英¹，　张琳¹，　董为人¹，　牛红心²，　王海红¹

作者信息 +

Application of Ara-C in rat hippocampal neuron purificationculture

LI Ang¹, YAN Fang¹, LI Zhen-lin¹, LIU Zhong-ying¹, ZHANG Lin¹, DONG Wei-ren¹, NIU Hong-xin², WANG Hai-Hong¹

Author information +

文章历史 +

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。

Abstract

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.

导出引用

李昂,晏芳,李振林,刘忠英,张琳,董为人,牛红心,王海红. 探讨阿糖胞苷在原代大鼠海马神经元培养中的纯化方法[J]. 中国临床解剖学杂志. 2015, 33(6): 681-684 https://doi.org/10.13418/j.issn.1001-165x.2015.06.014

Application of Ara-C in rat hippocampal neuron purificationculture[J]. Chinese Journal of Clinical Anatomy. 2015, 33(6): 681-684 https://doi.org/10.13418/j.issn.1001-165x.2015.06.014

参考文献

[1] Dotti CG， Sullivan CA， Banker GA. The establishment of polarity by hippocampal neurons in culture[J]. J Neurosci, 1988, 8(4): 1454-1468.

[2] Bosnjak M，Ristic B， Arsikin K，et al. Inhibition of mTOR-dependent autophagy sensitizes leukemic cells to cytarabine-induced apoptotic death[J]. PLoS One, 2014, 9(4): e94374.
[3] Xu Y， Cao C， Gong X，et al. Inhibition of ERK5 enhances cytarabine-induced apoptosis in acute myeloid leukemia cells[J]. Int J Clin Exp Med, 2015, 8(4): 6446-6455.
[4] Li G， Yan W， Dang Y，et al. The role of calcineurin signaling in microcystin-LR triggered neuronal toxicity[J]. Sci Rep, 2015, 5: 11271.
[5] 宋岳涛, 洪庆涛, 唐一鹏. 阿糖胞苷对原代培养的大鼠大脑皮层神经元的影响[J]. 解剖学杂志, 2004, 27(6): 696-699.
[6] 尹金宝, 常全忠, 韩宇东, 等. 新生大鼠离体海马神经元原代培养方法及鉴定[J]. 神经解剖学杂志, 2013, 28(6): 613-616.
[7] Ammendrup-Johnsen I， Naito Y， Craig AM，et al. Neurotrophin-3 enhances the synaptic organizing function of TrkC-protein tyrosine phosphatase sigma in rat hippocampal neurons[J]. J Neurosci, 2015, 35(36): 12425-12431.
[8] Yoshikawa K. Cell cycle regulators in neural stem cells and postmitotic neurons[J]. Neurosci Res, 2000, 37(1): 1-14.
[9] Raut LS. Novel formulation of cytarabine and daunorubicin: A new hope in AML treatment[J]. South Asian J Cancer, 2015, 4(1): 38-40.
[10]Lu JW， Lin YM， Lai YL，et al. MK-2206 induces apoptosis of AML cells and enhances the cytotoxicity of cytarabine[J]. Med Oncol, 2015, 32(7): 650.
[11]白雪涛. 利用胶质细胞滋养层进行分离式原代神经元培养[J]. 卫生研究, 2002, 31(4): 299-300.
[12]曾可斌，胡长林，陈阳美. 大鼠海马神经元培养与鉴定[J]. 基础医学与临床, 2004, 24(5): 574-577.
[13]Shafit-Zagardo B， Kalcheva N. Making sense of the multiple MAP-2 transcripts and their role in the neuron[J]. Mol Neurobiol, 1998, 16(2): 149-162.
[14]Desai UN， Shah KP， Mirza SH，et al. Enhancement of the cytotoxic effects of Cytarabine in synergism with Hesperidine and Silibinin in Acute Myeloid Leukemia: An in-vitro approach[J]. J Cancer Res Ther, 2015, 11(2): 352-357.