基于细胞培养基中血清水平优化探索赭曲霉毒素A对HK-2细胞的毒性作用  

Exploration of the toxic effects of ochratoxin A on HK-2 cells by optimizing serum level in cell culture medium

作　　者：董芳 张志岐 聂冬霞[2] 范楷[2] 娄秀萍 朱雪婷 韩铮[2] 赵志辉[2] DONG Fang;ZHANG Zhiqi;NIE Dongxia;FAN Kai;LOU Xiuping;ZHU Xueting;HAN Zheng;ZHAO Zhihui(College of Food Science,Shanghai Ocean University,Shanghai 201306,China;Institute for Agro-food Standards and Testing Technology,Shanghai Academy of Agricultural Sciences,Shanghai 201403,China)

机构地区：[1]上海海洋大学食品学院,上海201306 [2]上海市农业科学院农产品质量标准与检测技术研究所,上海201403

出　　处：《南京农业大学学报》2025年第1期220-229,共10页Journal of Nanjing Agricultural University

基　　金：上海市科技兴农项目(沪农科创字〔2021〕第3-2号)。

摘　　要：[目的]本文旨在探讨细胞培养基中添加不同水平血清对赭曲霉毒素A(ochratoxin A,OTA)毒性的影响,以期更深入了解OTA的毒性,同时为其他OTA体外毒性研究提供重要参考。[方法]测定OTA对人肾皮质近曲小管上皮细胞(human proximal tubule-derived epithelial cells,HK-2)细胞活力的影响,同时利用超高效液相色谱-串联质谱技术检测细胞上清液中OTA浓度以间接反应细胞对OTA的摄入量,分析培养基中不同水平血清对OTA毒性的影响。[结果]细胞培养基中添加5%的血清在48 h内不会影响细胞活力,可以作为细胞正常培养条件;添加2%的血清在36 h不会影响细胞活力,可以作为配制OTA工作液的细胞处理条件。基于优化的细胞培养及处理条件,OTA对HK-2细胞活力的影响结果表明,OTA抑制细胞活力呈现时间和剂量依赖性,OTA处理HK-2细胞12、24、36 h的IC50分别为39.29、6.28和2.72 μmol·L^(-1)。同时OTA可以时间依赖性极显著提高细胞释放乳酸脱氢酶水平,并损伤细胞膜的完整性(P<0.01)。[结论]细胞培养基中添加不同水平的血清会影响OTA的毒性,且血清水平越高,对OTA毒性影响越大。当使用2%血清的培养基配制OTA工作液时,可在不影响细胞活力的前提下最大限度降低血清水平对OTA毒性的影响。[Objectives]This paper aimed to investigate the effects of cell culture medium adding different levels of serum on ochratoxin A(OTA)toxicity to understand the toxicity of OTA better,and to provide an important reference for other studies in vitro. [Methods]The effect of OTA on the cell viability of human proximal tubule-derived epithelial cells(HK-2)was firstly determined,and OTA concentration in the cell supernatant was detected by ultra-high-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)in order to indirectly respond to the cellular uptake of OTA. The effect of adding different levels of serum in the culture medium on OTA toxicity was analyzed. [Results]The addition of 5% serum to the cell culture medium didn't affect the cell viability within 48 h and could be used as a normal cell culture condition;the addition of 2% serum didn't affect the cell viability at 36 h and could be used as a cell processing condition for the preparation of OTA working solution. Based on the optimized cell culture and treatment conditions,the effect results of OTA on the viability of HK-2 cells showed that the inhibition of OTA on HK-2 cells was time-dependent and dose-dependent. The IC50 of HK-2 cells treated by OTA for 12,24 and 36 h was 39.29,6.28 and 2.72 μmol·L^(-1),respectively. Meanwhile,OTA could also significantly increase the release level of lactate dehydrogenase in a time-dependent manner and damage the integrity of the cell membrane(P<0.01). [Conclusions]The toxicity of OTA could be affected by the serum in the cell medium,and the higher the serum level,the greater the impact on OTA toxicity. When OTA working solutions was prepared using media with a 2% serum,the effect of serum level on OTA toxicity could be minimized without affecting cell viability.

关 键 词：赭曲霉毒素A 血清水平 HK-2细胞 超高效液相色谱-串联质谱技术 

分 类 号：R994.4[医药卫生—毒理学]