南方医科大学学报

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (4): 617-626.doi: 10.12122/j.issn.1673-4254.2024.04.01

• •    下一篇

氧化石墨烯纳米颗粒在小鼠骨骼肌和人血液中诱导的免疫反应与毒性效应

孙益明，黄爱兰，赵 志，宋 晨，赖桂华   

  1. 蚌埠医科大学人体解剖学教研室，食品质量与安全教研室，第一附属医院骨科，安徽 蚌埠 233030；南方医科大学人体解剖学教研室，广东省组织工程构建与检测重点实验室，广东 广州 510515
  • 发布日期:2024-04-29

Immunogenic and toxic effects of graphene oxide nanoparticles in mouse skeletal muscles and human red blood cells

SUN Yiming, HUANGAilan, ZHAO Zhi, SONG Chen, LAI Guihua   

  1. Department of Human Anatomy, Department of Food Quality and Safety, Department of Orthopedics of First Affiliated Hospital, Bengbu Medical University, Bengbu 233030, China; Department of Anatomy, Guangdong Provincial Key Laboratory of Construction and Detection in Tissue Engineering, Southern Medical University, Guangzhou 510515, China
  • Published:2024-04-29

RichHTML

65

PDF

359

摘要： 目的 探讨氧化石墨烯（GO）在小鼠骨骼肌和血液中的免疫反应性，评估其毒性效应。方法 超声破碎法制备GO纳米颗粒，动态光散射仪测定悬浮于去离子水和PBS的GO纳米颗粒粒径与表面电荷。将不同浓度（0.5、1和2 mg/mL）的GO悬浮液或PBS分别注入C57BL/6小鼠腓肠肌，HE和免疫荧光染色评价小鼠体内炎症和免疫反应性。采用扫描电镜、分光光度计和血栓弹性描记图（TEG）观察GO对体外人血红细胞形态、溶血和凝血的影响。结果 动态光散射研究显示，与水悬浮液相比，GO颗粒的PBS悬浮液具有更优的胶体分散性、稳定性、表面电荷效应及体内微环境模拟性。HE和免疫荧光染色显示，小鼠腓肠肌中GO植入区周围的炎症浸润、肌纤维变性以及巨噬细胞、单核细胞、树突状细胞和 CD4+ T 细胞的浸润程度（包括F4/80+、CD11b+、CD11c+的平均阳性荧光面积百分比和 CD3+CD4+细胞数等）均明显高于对照组（P<0.05），且呈明显的剂量和时间依赖性。扫描电镜观察显示，相比低浓度（0.002和0.02 mg/mL）的GO和PBS，较高浓度（0.2、2 和 20 mg/mL）的GO可使体外红细胞形态发生明显改变并引起红细胞溶血（P<0.05）。TEG 显示，相较低浓度组和PBS组，高浓度GO组的多项血液凝固参数（K, α, R和MA）出现明显异常。结论 GO可持久诱导炎症和免疫反应性，因而具有一定的骨骼肌肌肉毒性和体外血液毒性。

关键词: 氧化石墨烯；免疫反应；肌肉毒性；血液毒性

Abstract: Objective To investigate immunogenic and toxic effects of graphene oxide (GO) nanoparticles in mouse skeletal muscles and in human blood in vitro. Methods GO nanoparticles prepared using a probe sonicator were supended in deionized H2O or PBS, and particle size and surface charge of the nanoparticles were measured with dynamic light scattering (DLS). Different concentrations (0.5, 1.0 and 2.0 mg/mL) of GO suspension or PBS were injected at multiple sites in the gastrocnemius muscle (GN) of C57BL/6 mice, and inflammatory response and immune cell infiltrations were detected with HE and immunofluorescence staining. We also examined the effects of GO nanoparticles on human red blood cell (RBC) morphology, hemolysis and blood coagulation using scanning electron microscope (SEM), spectrophotometry, and thromboelastography (TEG). Results GO nanoparticles suspended in PBS exhibited better colloidal dispersity, stability and surface charge effects than those in deionized H2O. In mouse GNs, injection of GO suspensions dose- and time-dependently resulted in sustained muscular inflammation and myofiber degeneration at the injection sites, which lasted till 8 weeks after the injection; immunofluorescence staining revealed obvious infiltration of monocytes, macrophages, dendritic cells and CD4+ T cells around the injection sites in mouse GNs. In human RBCs, incubation with GO suspensions at 0.2, 2.0 and 20 mg/mL, but not at 0.002 or 0.02 mg/mL, caused significant alterations of cell morphology and hemolysis. TEG analysis showed significant abnormalities of blood coagulation parameters following treatment with high concentrations of GO. Conclusion GO nanoparticles can induce sustained inflammatory and immunological responses in mouse GNs and cause RBC hemolysis and blood coagulation impairment, suggesting its muscular toxicity and hematotoxicity at high concentrations.

Key words: graphene oxide; immune response; muscular toxicity; hematotoxicity