饲料及原料中赭曲霉毒素A污染普遍存在，严重危害畜禽健康。其具有免疫毒性，可通过DNA损伤及诱导的细胞周期阻滞与凋亡，影响免疫细胞活性。申请者前期研究发现，赭曲霉毒素A可诱导巨噬细胞ANA-1发生S期阻滞，导致细胞凋亡，但其引起S期阻滞的分子机制仍有待阐明。本项目在前期工作基础上，利用彗星实验、有丝分裂指数分析等进一步研究赭曲霉毒素A引起ANA-1细胞DNA损伤作用，采用流式细胞、免疫共沉淀、Western Blot、阻断剂阻断和siRNA干扰等方法，研究赭曲霉毒素A对Cdk2、CyclinE、CyclinA等S期调节蛋白表达水平的影响，重点针对DNA损伤检查点关键信号分子，研究ATM/ATR-Chk1/Chk2信号通路激活情况，探讨赭曲霉毒素A造成细胞DNA损伤并诱发S期阻滞的分子机制，以期深化对赭曲霉毒素A免疫毒性机制的理解，为畜禽生产中霉菌毒素防控策略及安全限量研究提供参考。

Ochratoxin A (OTA) is a ubiquitous mycotoxin in feedstuffs and raw materials with potential immunotoxic effects. DNA damage and cell phase arrest are the main pathway of the cytotoxicity induced by mycotoxins. According to our previous research, OTA could induce S phase arrest and apoptosis in mouse macrophage ANA-1 cells in vitro. However, the exact mechanism of S phase arrest induced by OTA remained unknown. To investigate the potential molecular mechanisms of S phase arrest induced by OTA in mouse macrophage ANA-1 cells, we are preparing to confirm the DNA damage by comet assay and mitotic index analysis. And then, the levels of Cdk2, CyclinE and CyclinA, which are the main factors involved in the S phase control and influenced by OTA in ANA-1, will be evaluated by using flow cytometric analysis, Western Blot, co-immunoprecipitation. Based on the already-know factors involved in the DNA damage checkpoint, we will analyzing the activation of ATM/ATR-Chk1/Chk2 pathway to distinguish the role of ATM, ATR, Chk1, Chk2, Cdc25A and Wee1 in S phase checkpoint after OTA-induced DNA damage, by using flow cytometric analysis, Western Blot, co-immunoprecipitation, inhibitors and siRNA transfection. This research will elucidated the potential molecular mechanisms of DNA damage and S phase arrest induced by OTA in mouse macrophage ANA-1 cells in vitro. The results of this research will contribute to understand the mechanism of immunotoxic effects of OTA, and provide the basic evidences for mycotoxin control and maximum levels set in animal husbandry.