呕吐毒素损伤猪肠道干细胞发育的机制及营养干预

Deoxynivalenol (DON) contamination of feed based on grain causes serious threat to swine industry by injury intestinal epithelium structure and function. However, it is not clear whether it damages the development of intestinal stem cells. . On the basis of our preliminary studies, the object of this proposal will be firstly to determine the sensitivity of porcine intestinal stem cell to DON by acute/chronic toxicity treatment in vivo. The effect of DON on the stem cell development and the epithelium renewal of pig intestine will be investigated through Immunohistochemistry (fluorescence) and flow cytometry. Meanwhile, the activity of Wnt/β-catenin signaling pathway in the niche of intestinal stem cell after DON-treated is analyzed via Wes protein expression quantitative system. Moreover, β-catenin over expression or knockdown of porcine intestinal stem cell three dimensional (3D) culture system in vitro will be constructed using lentivirus infection technology, to explore the molecular mechanism of intestinal stem cell development injury induced by DON mediated by Wnt/β-catenin signal. Finally, To validate whether nutritional intervention can attenuate the intestinal stem cell injury caused by DON and probe into its rescue mechanism, dietary or stem cell culture medium are supplemented with methionine/methionine hydroxy analogue (HMB), in feeding experiment or intestinal stem cell 3D culture in vitro, respectively. . In summary, the results not only clarify the molecular mechanism of intestinal injury induced by DON from the point of stem cell for the first time, but also provide evidence for developing nutritional manipulation technology in safety and high efficiency.

饲料源呕吐毒素可导致肠上皮结构受损，危害养猪业，但其是否通过损伤肠道干细胞发育发挥作用尚不清楚。本项目在前期基础上，首先通过活体急/慢性和离体攻毒试验，确定猪肠道干细胞对呕吐毒素的敏感性；借助免疫组化(荧光)和流式细胞技术，研究呕吐毒素对猪肠道干细胞发育和肠上皮更新的影响；采用Wes蛋白质表达定量系统，分析呕吐毒素损伤刺激，导致猪肠道干细胞微环境中Wnt/β-catenin信号变化规律。然后利用慢病毒感染技术，调节猪肠道干细胞β-catenin的表达，探究Wnt/β-catenin信号介导呕吐毒素损伤猪肠道干细胞发育的分子机制。最后通过饲养试验、肠道干细胞体外培养试验和营养干预，在日粮或干细胞培养液中添加蛋氨酸/蛋氨酸羟基类似物，探明其逆转呕吐毒素诱导猪肠道干细胞发育损伤的作用机理。研究结果不仅在理论上首次从干细胞角度，揭示呕吐毒素作用的分子机制，还为研发安全、高效的营养调控技术提供依据。

呕吐毒素是谷物籽实和饲料中污染最广泛的霉菌毒素，猪是对呕吐毒素最敏感的动物，肠道是呕吐毒素发挥毒性的首要靶点，但其是否通过损伤肠道干细胞活性发挥作用尚不清楚。本项目以仔猪和肠道类器官为模型，综合运用营养学、细胞生物学、分子生物学和蛋白质组学等技术手段，从干细胞发育、信号网络筛查、信号分子反向验证及营养素干预四个部分研究内容系统解析呕吐毒素的作用机制。结果表明：1）急慢性呕吐毒素均破坏仔猪小肠结构和屏障功能，抑制肠道干细胞体外扩增；2）急慢性呕吐毒素下调仔猪空肠和隐窝中Wnt/β-catenin信号通路；3）重组表达的Wnt/β-catenin激动剂R-spondin 1能逆转呕吐毒素暴露下仔猪小肠和类器官损伤；4）蛋氨酸及其羟基类似物可激活Wnt/β-catenin信号，修复呕吐毒素暴露下受损的小肠干细胞。该研究确认了呕吐毒素扰乱肠道干细胞增殖分化命运，阐明了Wnt/β-catenin信号通路响应呕吐毒素损伤干细胞的分子机制，构建了以蛋氨酸及其羟基类似物为核心的营养干预方案。已有数据发表SCI论文15篇（第一资助6篇），申请发明专利3项（授权1项），培养研究生5名（博士1名，硕士4名）。该项目结果为饲料源呕吐毒素防治提供了理论指导。