氧化应激和Wnt/β-catenin信号通路在赭曲霉毒素A诱导胃黏膜上皮细胞恶性转化中的作用及意义

Mycotoxin contamination in food was closely correlated with the occurrence of human diseases. Ochratoxin A (OTA) has been classified as "possibly carcinogenic to humans". Our previous study showed that high levels of OTA contamination in wheat have been detected in the high incidence area of gastric carcinoma in China. At the same time, studies in vitro have found that short-term exposure of OTA could induce oxidative stress damage, while long-term exposure induce invasion in human gastric epithelial cell line (GES-1). It has been generally accepted that oxidative stress damage was an important factor in tumorigenesis. However, there was no available data regarding the relation between OTA-induced oxidative stress damage and gastric carcinogenesis up to now. In the present study, we explored the biological characteristics and invasion of GES-1 cells after long-term exposure of OTA by Transwell method. We further explored the effect of Wnt/β-catenin signaling pathway in the process of OTA-induced malignant transformation in GES-1 cells by Western blot. In addition, based on the implementation of antioxidant strategies, the effect of oxidative stress damage in Wnt/β-catenin signaling pathway was also evaluated in this study. Finally, we observed the damage effect of OTA on gastric epithelial cell from the whole level by the establishment of animal model, and to provide scientific basis for revealing the possible relationship between OTA and gastric carcinogenesis.

饮食中真菌毒素的污染与人类疾病的发生关系密切。赭曲霉毒素A（OTA）是"可能的人类致癌物"。申请者前期发现我国胃癌高发区居民食用小麦中OTA的污染严重，体外研究发现短期暴露OTA可致人胃黏膜上皮细胞（GES-1)氧化应激损伤；而长期暴露OTA可使细胞获得侵袭性。氧化应激被认为是启动和促进肿瘤发生的最重要因素。但目前有关OTA所致的氧化应激损伤与胃癌发生关系的研究未见报道。本研究首先利用Transwell迁移等技术探讨长期暴露OTA对GES-1细胞生物特性以及侵袭性的影响；接着以Wnt/β-catenin通路作为切入点，利用蛋白印迹等技术揭示Wnt通路在OTA诱导GES-1细胞恶性转化中的作用；然后基于抗氧化策略的实施，评价氧化应激对Wnt/β-catenin通路的影响；最后建立动物模型，从整体水平观察OTA对胃粘膜上皮细胞的损伤作用，为揭示OTA与胃癌发生的关系提供科学依据。

饮食中真菌毒素的污染与人类疾病的发生关系密切。赭曲霉毒素A（OTA）是“可能的人类致癌物”。申请者前期调查发现我国胃癌高发区居民食用小麦中OTA的污染严重，体外研究发现短期暴露于OTA可致人胃黏膜上皮细胞（GES-1)氧化应激损伤和细胞周期紊乱。本项目在前期研究的基础上，通过延长OTA的暴露时间，探讨OTA对GES-1细胞氧化应激损伤、上皮-间质转化及恶性转化之间的关系及分子机制，揭示OTA的高污染与胃癌发生的可能关系。.本项目研究结果显示，OTA长期处理人胃黏膜上皮细胞可以增加上皮标记的表达，同时降低间质标记的表达，导致GES-1细胞发生上皮-间质转化。研究还显示OTA长期处理GES-1细胞可以增加细胞的迁移力和侵袭力，并利用该转化细胞株成功建立了裸鼠成瘤模型。OTA恶性转化人胃粘膜上皮细胞株以及裸鼠成瘤模型的成功建立在国内外尚属首次。此外，我们的研究还显示OTA长期处理GES-1细胞可以激活WNT信号通路。给予WNT信号通路的抑制剂DKK-1预处理GES-1细胞后，结果显示DKK-1可以抑制OTA诱导的上皮-间质转化。同时，进一步给予抗氧化剂NAC预处理GES-1细胞后，结果显示抗氧化剂NAC可以抑制OTA长期暴露诱导的WNT信号通路的激活、抑制GES-1细胞EMT及恶性转化的发生。以上研究结果表明，OTA长期暴露GES-1细胞后可以激活WNT信号通路，诱导上皮-间质转化及细胞恶性转化的发生，而在这个过程中氧化应激损伤发挥了至关重要的作用。最后，我们将OTA灌胃小鼠进行体外实验，研究初步发现OTA灌胃5个月后小鼠食欲明显下降，同时胃粘膜上皮细胞发生氧化应激损伤。本项目的研究成果对胃癌高发区胃癌病因的研究具有非常重要的意义，同时也积极推动了真菌毒素致癌性研究的发展。