基于HaCaT三维表皮模型的纳米银毒性及其毒性机制研究

目的：应用三维（3D）表皮模型（HaCaT模型，无角质层），与二维（2D）HaCaT细胞对比，评价等效浓度下纳米银（AgNPs）对HaCaT模型的毒性效应，并考察其毒性机制。方法：选用同等浓度，即：单位细胞数AgNPs暴露浓度（HaCaT模型组为62.5、125、250、500、1 000 μg·mL^-1，对应于HaCaT细胞组为1.75、3.5、7、14、28 μg·mL^-1），分别暴露于HaCaT模型及单层HaCaT细胞24 h后，用噻唑盐比色法（MTT）和乳酸脱氢酶释放试验（LDH）测定AgNPs在HaCaT模型和HaCaT细胞中的毒性效应。取同等AgNPs浓度（单位细胞0.7 ng·mL^-1）暴露组，用流式细胞仪检测模型或细胞中活性氧（ROS）含量的变化；用相应试剂盒检测模型或细胞中超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）的活性及丙二醛（MDA）的含量；用酶联免疫吸附试验（ELISA）检测模型或细胞培养液中的炎性因子IL-1α、TNF-α、IL-6和IL-8的含量。结果：随着AgNPs浓度的增加，MTT实验显示HaCaT模型相对组织活力下降至（91.16±3.59）%~（75.66±1.66）%，而HaCaT细胞活性下降至（77.42±5.17）%~（22.43±2.48）%，二者均呈剂量-效应关系；LDH实验显示HaCaT模型的LDH释放量从（0.17±1.12）%升高至（44.9±3.61）%，而HaCaT细胞的LDH释放量从（55.99±1.58）%升高至（68.68±1.42）%，二者均呈剂量-效应关系。AgNPs作用于HaCaT模型和细胞后，均引起SOD和GSH-px合成量下降，ROS的释放量及MDA升高（分别为对照组的3.7倍和11.5倍）；抗氧化剂（NAC）能够不同程度地减少以上氧化应激因子的变化；引起IL-1α释放量显著升高（分别为对照组的2.97倍和11.55倍），IL-6和IL-8的释放量降低（分别为对照组的2.09倍和1.36倍、1.4倍和3.83倍）。结论：HaCaT模型较好地模拟了创伤皮肤的形态结构，能够客观地评价创伤皮肤外用敷料的局部作用，是评价纳米材料皮肤毒性风险的候选替代模型。

Objective: To evaluate the toxic effect and its mechanisms of silver nanoparticles (AgNPs) at equivalent exposure concentration on three-dimensional (3D) epidermal model (HaCaT model without cuticle),compared with two-dimensional (2D) HaCaT cells. Methods: The equivalent concentration (HaCaT model group was set as 62.5,125,250,500,1 000 μg·mL^-1,corresponding to 1.75,3.5,7,14,28 μg·mL^-1 in HaCaT cell group) were exposed to HaCaT model and HaCaT cells for 24 hours,and the toxic responses were assessed by thiazolyl[3-(4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-H-bromide] ratio (MTT) assay and lactate dehydrogenase (LDH) release assay;In the exposure group with the same AgNPs concentration (0.7 ng·mL^-1 per cell),equivalent concentration,the changes of reactive oxygen species (ROS) content in the model or cells were detected by flow cytometry. The activity of superoxide dismutase (SOD),glutathione peroxidase (GSH-Px) and the content of Malondialdehyd (MDA) in the model or cells were detected with the corresponding kit. The levels of inflammatory cytokines IL-1α、TNF-α、IL-6和IL-8 in the model or cell culture solution were detected by ELISA. Results: With the increase of AgNPs exposure concentration,the MTT assay showed that the relative tissue viability of HaCaT model decreased from (91.16±3.59)% to (75.66±1.66)%,while the relative cell viability of HaCaT cells decreased from (77.42±5.17)% to (22.43±2.48)%,both of which showed a dose-dependent manner. The LDH assay showed that the LDH release in HaCaT model increased from (10.17± 1.12)% to (44.9±3.61)%,while the LDH release in HaCaT cells increased from (55.99±1.58)% to (68.68±1.42)%,both of which showed a dose-dependent manner. After AgNPs exposed on HaCaT model (500 μg·mL^-1) and cells (14 μg·mL^-1),the synthesis of SOD and GSH-px were decreased,while the release of ROS and MDA (3.7-times and 11.5-times compared to control,respectively) were increased;and antioxidant NAC can decrease the changes of these oxidative stress factors;and the release of IL^-1a increased (2.97 times and 11.55 times compared to control,respectively),but the release of IL-6 and IL-8 were decreased. Conclusion: HaCaT model can simulate the morphological structure of wounded skin and objectively evaluate the local effects of wound dressings. It is a candidate alternative model for evaluating the skin toxicity risk of nanomaterials.

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