石墨碳纳米颗粒对体外培养细胞生长特性的影响

背景：有报道指出石墨碳纳米颗粒具有强大的吸附能力，只要尽可能将其控制在有效浓度范围内，石墨碳纳米颗粒会具有很好的细胞相容性及增敏效应。目的：了解石墨碳纳米颗粒的形态特征，观察石墨碳纳米颗粒其对体外培养细胞增殖与超微结构的影响。方法：取石墨碳纳米颗粒0.5 g，加100 mL三蒸水，振荡后微孔过滤，即为石墨碳纳米颗粒母液。取处于对数生长期的HepG2细胞、L02细胞、Hl7702细胞、3T3细胞，调整密度为5×107 L-1接种于6孔板，0.5 mL/孔，加入含胎牛血清、青霉素、链霉素的RPMI-1640培养基1.5 mL，培养24 h后弃去旧液，设1~5号孔为实验组，分别加入质量浓度为25，10，7.5，5，0.25 mg/L石墨碳纳米颗粒培养液2.0 mL，设6号孔为空白对照组，不加石墨碳纳米颗粒溶液，继续培养24 h后终止培养。用原子力显微镜测量石墨碳纳米颗粒的粒径，电子显微镜观察石墨碳纳米颗粒的形态特征；用细胞计数板在光学显微镜下计数不同浓度石墨碳纳米颗粒对细胞数量的影响；透射电镜观察7.5 mg/L石墨碳纳米颗粒对细胞超微结构的影响。结果与结论：石墨碳纳米颗粒呈球形微粒，粒径约20 nm。与空白对照组比较，各浓度石墨碳纳米颗粒培养液组除HepG2细胞外，其余3种细胞数量基本都有所增加，其中7.5 mg/L石墨碳纳米颗粒培养液对L02细胞、Hl7702细胞、3T3细胞、HepG2细胞数量的影响最为显著(P < 0.05)。对7.5 mg/L石墨碳纳米颗粒作用后的细胞进行透射电镜观察，可见石墨碳纳米颗粒分布于细胞内部，如细胞质、细胞核、线粒体中，未见亚细胞结构受损及细胞凋亡坏死现象发生。证实石墨碳纳米颗粒对体外培养的细胞无损伤不良反应，且能够促进细胞生长增殖，其作用强度与质量浓度有关，7.5 mg/L为较佳质量浓度。关键词：纳米石墨碳；人肝细胞株；人肝癌细胞株；超微结构；细胞生长doi:10.3969/j.issn.1673-8225.2010.03.015

Effect of graphite carbon nanoparticles on cell growth in vitro

BACKGROUND: Previous research has indicated that graphite carbon nanoparticles have a strong adsorbability. While, when the concentration is effectively controlled, graphite carbon nanoparticles also have well compatibility and sensitizing effect. OBJECTIVE: To observe the morphology of graphite carbon nanoparticles, and to investigate the effects of graphite carbon nanoparticles on cell proliferation and ultramicrostructure.METHODS: Graphite carbon nanoparticles (0.5 g) were put in 100 mL triple distilled water to obtain graphite carbon nanoparticle mother liquid after oscillation and microfiltration. HepG2 cells, L02 cells, Hl7702 cells, and 3T3 cells in the logarithmic phase were adjusted to the concentration of 5×107/L and inoculated in 6-well culture plate with 0.5 mL per well. Thereafter, the cells were cultured with RPMI-1640 culture media (1.5 mL) containing fetal bovine serum, penicillin, and streptomycin. The original culture solution was removed after 24 hours. The 1-5 wells were considered as the experimental group, and 25, 10, 7.5, 5, 0.25 mg/L graphite carbon nanoparticles (2.0 mL) were respectively added into each well; while, the sixth well was considered as the blank control group without graphite carbon nanoparticles. The cells in the blank control group were cultured for 24 hours. Particle diameter was measured using atomic force microscopy; morphology was observed using electron microscope; effect of different concentrations of graphite carbon nanoparticles on cell number was detected using hemacytometry under optic microscope; the effect of 7.5 mg/L graphite carbon nanoparticles on ultramicrostructure was observed under transmission electron microscope. RESULTS AND CONCLUSION: Graphite carbon nanoparticles were around and 20 nm diameter. Compared with the blank control group, cell numbers except HepG2 cells were increased, especially the effect of 7.5 mg/L graphite carbon nanoparticles was greatest (P < 0.05). Transmission electron microscope indicated that graphite carbon nanoparticles were distributed into cells, including cytoplasm, nucleus, and mitochondrion; while, subcellular structure damage and cell apoptosis and necrosis were absent. Graphite carbon nanoparticles have no side effects on in vitro cultured cells and can promote cell proliferation, showing a dose-dependence correlation, especially the concentration of 7.5 mg/L.