目的：通过构建体内和体外染毒模型，观察氧化锌纳米颗粒(ZnO NPs)对角膜的毒性影响。方法：体外培养原代人角膜上皮细胞(HCEpiC)，暴露于0.5、5、12.5、25、50、100、250μg/mL氧化锌纳米颗粒24h，设不含纳米溶液的细胞培养液为空白对照组。采用MTT比色法评估细胞的活性。三种不同浓度(25、50和100μg/mL)的氧化锌纳米颗粒分散液暴露于麻醉后小鼠结膜囊内，磷酸盐平衡缓冲液(PBS)点眼为PBS对照组，每天3次，连续7d，第1、3、5、7d观察角膜形态，第8d取出眼球，观察角膜病理改变和炎症因子(TNF-α，IL-6)表达水平情况。结果：不同浓度的氧化锌纳米颗粒处理原代人角膜上皮细胞24h后，MTT结果显示，与空白对照组相比，氧化锌纳米颗粒在0.5μg/mL即对细胞有损伤作用，细胞存活率约80%(P<0.05)，5μg/mL剂量时可致半数细胞死亡，在5～250μg/mL浓度范围对细胞的损伤作用具有浓度依赖性(P<0.0001)。不同浓度的氧化锌纳米颗粒暴露小鼠结膜囊，点眼7d后，25μg/mL和50μg/mL氧化锌纳米颗粒组可见部分角膜上荧光素的点状着染，100μg/mL氧化锌纳米颗粒组角膜见局部圆形荧光素着染区。HE染色结果显示，25μg/mL和50μg/mL氧化锌纳米颗粒组的角膜上皮层、基质层厚度、基质层免疫细胞数目无明显改变(均P>0.05)，而100μg/mL氧化锌纳米颗粒组角膜上皮层变薄、角膜基质层变厚、基质层免疫细胞明显增多(均P<0.05)。免疫组织化学染色结果显示，100μg/mL氧化锌纳米颗粒组产生TNF-α、IL-6的角膜基质免疫细胞数及TNF-α、IL-6的平均光密度(IOD)值均明显高于PBS对照组(P<0.05)，且炎症反应程度具有浓度依赖性，25μg/mL氧化锌纳米颗粒和50μg/mL氧化锌纳米颗粒组免疫细胞数和IOD值与PBS对照组相比均无明显增高(P>0.05)。结论：氧化锌纳米颗粒在体外和体内均对角膜具有毒性损伤作用，为氧化锌纳米颗粒眼部安全性评价提供了一定的理论依据。

AIM:To observe the toxic effects of zinc oxide nanoparticles(ZnO NPs)on cornea by constructing intoxicated model in vivo and in vitro.METHODS:Human corneal epithelial cells(HCEpiC)were cultured in vitro and exposed to different concentrations(0.5, 5, 12.5, 25, 50, 100, 250 μg/mL)of ZnO NPs for 24h. The cell culture medium without nano-solution was used as the blank control group. The viability of the cells was assessed by MTT assay. Three different concentrations(25, 50 and 100 μg/mL)of ZnONPs dispersions were exposed to the conjunctival sac of anesthetized mice three times a day for 7d consecutively. The phosphate buffered saline(PBS)eye group was the PBS control group. Corneal morphology was observed on 1, 3, 5 and 7d, and the eyes were removed on 8d for various laboratory examinations, including corneal pathological changes and expression levels of inflammatory factors(TNF-α, IL-6).RESULTS:After treatment of HCEpiC cells with different concentrations of ZnO NPs for 24h, the MTT results showed that Zno NPs cause damage to cells at 0.5 μg/mL, and the cell survival rate was about 80%(P<0.05). Half of the cells were killed at a dose of 5 μg/mL, the damaging effect on cells in the concentration range of 5～250 μg/mL was concentration-dependent(P<0.0001). After 7d of conjunctival capsule spotting in mice, dot-like staining of fluorescein was seen in the 25 μg/mL ZnO NPs and 50 μg/mL ZnO NPs groups. Localized circular fluorescein stained areas were seen in the corneas of the 100 μg/mL ZnO NPs group. HE staining showed that the corneal epithelial layer, stromal layer thickness and stromal layer immune cell number did not change significantly in the 25 μg/mL and 50 μg/mL ZnO NPs groups(all P>0.05), while the corneal epithelial layer thinned, the corneal stromal layer thickened and the stromal layer immune cells increased significantly in the 100 μg/mL ZnO NPs group(all P<0.05). Immunohistochemical staining showed that the number of corneal stromal immune cells producing TNF-α and IL-6 and the mean integral optical density(IOD)values of TNF-α and IL-6 were significantly higher in the 100 μg/mL ZnO NPs group than in the PBS control group(P<0.05), and the degree of inflammation response was concentration-dependent. Compared with the PBS control group, no significant increase in immune cell count and IOD values in the 25 μg/mL ZnO NPs and 50 μg/mL ZnO NPs groups(P>0.05).CONCLUSION:The toxic damaging effect of ZnO NPs on the cornea was confirmed from both in vitro and in vivo, which provided a theoretical basis for the ocular safety evaluation of ZnO NPs.

国家重点研发计划“政府间国际科技创新合作”重点专项立项项目(No.2019YFE0117800); 国家自然科学基金资助项目(No.22176115)