氧化锌纳米颗粒在体内和体外对角膜的毒性评估  

Toxicity evaluation of zinc oxide nanoparticles in vitro and in vivo on cornea

作　　者：侯晓璐 崔冬梅 牛灵芝 孙晓彤 于涛 赵宇航 宋爱萍 李玮[2] Xiao-Lu Hou;Dong-Mei Cui;Ling-Zhi Niu;Xiao-Tong Sun;Tao Yu;Yu-Hang Zhao;Ai-Ping Song;Wei Li(Department of Ophthalmology,the First Affiliated Hospital of Shandong First Medical University(Shandong Provincial Qianfoshan Hospital),Jinan 250014,Shandong Province,China;School of Control Science and Engineering,Shandong University,Intelligent Medical Engineering Research Center,Shandong University,Jinan 250014,Shandong Province,China)

机构地区：[1]山东第一医科大学第一附属医院、山东省千佛山医院,中国山东省济南市250014 [2]山东大学控制科学与工程学院、山东大学智能医学工程研究中心,中国山东省济南市250014

出　　处：《国际眼科杂志》2023年第7期1080-1086,共7页International Eye Science

基　　金：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)。

摘　　要：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(0.5,5,12.5,25,50,100,250μg/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 100μg/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 0.5μg/mL,and the cell survival rate was about 80%(P<0.05).Half of the cells were killed at a dose of 5μg/mL,the damaging effect on cells in the concentration range of 5~250μg/mL was concentration-dependent(P<0.0001).After 7d of conjunctival capsule spotting in mice,dot-like staining of fluorescein was seen in the 25μg/mL ZnO NPs and 50μg/mL ZnO NPs groups.Localized circular fluorescein stained areas were seen in the corneas of the 100μg/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 25μg/mL and 50μg/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 100μg/mL ZnO NPs group(all P<0.05).Immunohistochemical staining showed that the number of corneal stromal immune cells producing TNF-αand IL-6 and the mean integral optical density(IOD)values of TNF-αand I

关 键 词：氧化锌纳米颗粒 角膜上皮细胞 小鼠 

分 类 号：R318.08[医药卫生—生物医学工程]