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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 400μg/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 400μg/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 400μg/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 400μg/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。
Objective: To investigate the effect and mechanism of gold nanoparticles (GNPs) with small diameter of 5 nm on rat cortical neurons. Methods: GNPs were prepared by reduction with sodium borohydride and characterized by transmission electron microscopy (TEM) and ultraviolet-visible spectroscopy (UV-Vis). GNPs culture medium was prepared by using 5% bovine serum albumin (BSA) to prepare GNPs culture medium with different concentrations (600, 1 200 and 2 400 μg / L). Primary cultures of rat cortical neurons were identified by immunofluorescence staining. Cultured primary rat cortical neurons 72 h later, the normal neurons were used as the control group, neurons in different concentrations of GNPs incubated for 48 h as the experimental group. The distribution of GNPs in cells was observed by TEM. Cell viability was detected by CCK-8 kit. Cell apoptosis was detected by TUNEL assay. Malondialdehyde (MDA) and superoxide dismutase (SOD) Level. Results: TEM and UV-Vis showed that the GNPs prepared by the sodium borohydride reduction method were uniform in size and uniformly dispersed in solution. Primary cultured rat cortical neurons were (82.0 ± 2.3)% pure. GNPs mainly in the form of polymer distribution in the cytoplasm, lysosomes, vesicles. With the increase of GNPs concentration, cell viability decreased gradually, especially at 1 200 and 2 400 μg / L, the cell viability was significantly lower than that of the control group (P <0.05). The results of TUNEL staining showed that with the increase of GNPs concentration, the number of apoptotic cells increased gradually, and the apoptosis rate was significantly different between 1 200 and 2 400 μg / L groups (P <0.05). With the increase of GNPs concentration, the content of MDA in the cells gradually increased and the content of SOD decreased gradually. Compared with the control group, the content of MDA and the content of SOD in 1 200 and 2 400 μg / L groups were significantly decreased (P <0.05). CONCLUSION: 5 nm GNPs can reduce the cell viability and promote the apoptosis of cortical neurons in rats, which may be related to the oxidative stress injury.