5 nm金纳米颗粒对大鼠皮质神经元的作用及机制初探
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国家自然科学基金(81300999,81470501)


Effects of 5 nm gold nanoparticles on rat cortical neurons and relative mechanism
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    摘要:

    目的:探讨直径5 nm的小尺寸金纳米颗粒(gold nanoparticles,GNPs)对大鼠皮质神经元细胞的作用及相关机制。方法:使用硼氢化钠还原法制备GNPs,用透射电镜(TEM)及紫外-可见光光谱(UV-Vis)进行鉴定。用5% 牛血清白蛋白(BSA)对GNPs进行包裹提纯后制备成不同浓度(600-1 200-2 400 μg/L)的GNPs培养液。采用免疫荧光染色鉴定原代培养的大鼠皮质神经元纯度。体外培养原代大鼠皮质神经元72 h后,以正常培养的神经元为对照组,以不同浓度GNPs溶液孵育48 h后的神经元作为实验组。采用TEM观察GNPs在细胞内的分布;采用CCK-8试剂盒检测细胞活力;采用TUNEL法检测细胞凋亡;采用丙二醛(MDA)-超氧化物歧化酶(SOD)试剂盒检测氧化应激水平。结果:TEM及UV-Vis显示硼氢化钠还原法制备的GNPs呈尺寸均一的球形,在溶液中均匀分散。原代培养的大鼠皮质神经元纯度为(82.0 ± 2.3)%。GNPs主要以聚合体形式分布于胞浆-溶酶体-囊泡中。随着GNPs浓度的增高,细胞活性逐渐降低,尤其1 200和2 400 μg/L处理组细胞活力较对照组显著降低(P < 0.05)。TUNEL染色结果显示,随着GNPs浓度的增高,凋亡细胞数逐渐增加,1 200和2 400 μg/L处理组细胞凋亡明显,具有显著性差异(P < 0.05)。随着GNPs浓度的增高,细胞内MDA含量逐渐增加,SOD含量逐渐减少,1 200和2 400 μg/L处理组与对照组相比MDA含量显著升高,SOD含量显著降低(P < 0.05)。结论:5 nm GNPs能降低大鼠皮质神经元的细胞活力,促进其凋亡,其机制可能与氧化应激损伤相关。

    Abstract:

    Objective:To investigate the effects of 5 nm gold nanoparticles(GNPs) on newborn rat cortical neurons and the underlying mechanism. Methods:GNPs were prepared by the NaBH4 reduction method. Transmission electron microscope(TEM) and ultraviolet-visible(UV-Vis) spectra were obtained to characterize GNPs. Immunofluorescence staining was performed to indentify primary neurons and calculate the purity. After culture for 72 h, primary cortical neurons were incubated with neuron culture medium with(600, 1 200, 2 400 μg/L) or without GNPs for 48 h. TEM was used to investigate the celluar uptake and distribution of GNPs; Cell counting kit-8(CCK-8) was employed to evaluate cell viability; TUNEL assay was performed to analyze apoptosis; MDA and SOD were measured to assess oxidative stress. Results:TEM and UV-Vis showed that GNPs were homogeneous in size and shape and well dispersed in culture medium. The purity of primary neurons was(82.7 ± 2.3)%. GNPs were mainly distributed in cytoplasm, lysosomes and vesicles. The cell viability was found to decrease with increasing concentrations of GNPs(P < 0.05). In addition, GNPs were found to induce apoptosis in neurons. Particularly, higher concentrations(1 200 μg/L and 2 400 μg/L) markedly increased neural apoptosis(P < 0.05). Furthermore, our results suggested pro-oxidant ability of 5 nm GNPs,which is supported by escalating MDA and reducing SOD along with increasing concentrations of GNPs(P < 0.05). Conclusion:Five nm GNPs can significantly reduce cell viability and induce apoposis in newborn rat cortical neurons, which potentially links to oxidative stress.

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吴玉芸,刘正霞,郭志睿,刘 莹,鲁 翔.5 nm金纳米颗粒对大鼠皮质神经元的作用及机制初探[J].南京医科大学学报(自然科学版),2016,(7):811-816

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  • 收稿日期:2015-10-28
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  • 在线发布日期: 2016-07-15
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