IFN-gamma 诱导奶牛乳腺上皮细胞精氨酸耗竭的分子机制

In recent years, it has been found that arginine depletion is a common phenomenon which associated with many diseases, and is also an important precipitating(regulatory)factor in pathogenesis. In addition to the regulation of immune cells, IFN-gamma can also induce the depletion of arginine in tissue cells, but its mechanism is still not clear. This project intends to use bovine mammary epithelial cells as a model, using transcriptomics, nuclear protein mass spectrometry and bioinformatics to analyze the possible pathway and signaling pathways of intracellular arginine metabolism and screen the key enzyme and protein molecules involved in arginine metabolism under IFN-gamma stimulation; using gene overexpression, RNA interference / gene knockout technique to verify the key nodes molecular and clarify the mutual regulatory interaction, to reveal the molecular mechanism of IFN-gamma induced arginine metabolism, recognition and identification of important molecular targets for manual intervention or treatment. The completion of this project not only comes up with the regulation theory of IFN-gamma to healthy cow breast tissue cells of arginine metabolism, provides new ideas for prevention of cow mastitis and milk quality improvement; but also lay the foundation for the thorough understanding of IFN-gamma induced by arginine metabolism disorders, enrich the knowledge of regulation of cytokines on tissue cells.

近年来发现精氨酸耗竭是多种疾病常见的伴发现象，也是发病机制的重要促发（调控）因素。IFN-gamma除对免疫细胞调控作用，能够诱导组织细胞发生精氨酸耗竭，但其机制并不清楚。本项目拟以奶牛乳腺上皮细胞为模型，采用转录组学、核蛋白质谱技术和生物信息学技术，解析IFN-gamma刺激下胞内精氨酸代谢可能的途径和信号通路，筛选IFN-gamma刺激下参与精氨酸代谢的关键酶和蛋白分子；通过基因过表达、RNA干扰/基因敲除术，确证信号通路中关键节点分子，明确调控关系，体外、体内揭示IFN-gamma诱导精氨酸代谢的分子机制，识别和鉴定可用于人工干预或临床治疗的重要分子靶标。该项目的完成不仅提出IFN-gamma对奶牛乳腺组织细胞精氨酸代谢调控理论，为奶牛乳房炎防治提供新思路；而且有助于深入理解精氨酸异常代谢相关病症的发生机理，丰富细胞因子对组织细胞的调控知识。

精氨酸是条件性必须氨基酸，具有多种重要的生理功能。近年来研究发现精氨酸耗竭与多种疾病的发生发展密切相关，因此阐明精氨酸耗竭机制十分必要。IFN-γ是种重要的促炎性细胞因子，除调控免疫外，IFN-γ长时间刺激能够诱导组织细胞发生精氨酸耗竭，但其机制并不清楚。本课题以奶牛乳腺上皮细胞（BMECs）为研究对象，采用代谢组学、转录组学、生物信息学和分子生物学技术，研究了IFN-γ对BMECs基本特性的影响，阐明了精氨酸代谢的关键分子和调控机制，鉴定可用于临床治疗的重要分子靶标。发现IFN-γ诱导的精氨酸耗竭促进BMECs 增殖和恶性变，抑制 BMECs 乳脂乳蛋白的合成，增强对S. aureus的易感性。代谢组学检测表明IFN-γ刺激可引起BMECs广泛的代谢异常，其中脂质代谢和氨基酸代谢受影响最为显著；IFN-γ通过促进精氨酸分解代谢和抑制其合成代谢，引起精氨酸耗竭。转录组学鉴定出肌肽合成酶CARNS1、亮氨酸氨基肽酶LAP3和半胱氨酸-tRNA合成酶CARS2是调控IFN-γ诱导的精氨酸耗竭和细胞恶性变的关键分子。IFN-γ通过与其受体IFNGR2结合，激活MAPKs信号通路调控CARNS1、CARS2和LAP3表达；CARNS1通过抑制精氨酸合成关键酶ASS1参与精氨酸代谢；CARS2通过抑制ASS1和鸟氨酸转氨甲酰酶OTC，下调瓜氨酸水平抑制精氨酸合成代谢，同时促进精氨酸脱羧酶ADC加速精氨酸分解代谢，介导精氨酸耗竭；LAP3同样通过抑制ASS1表达参与精氨酸耗竭，进一步通过组蛋白去乙酰化酶HDAC2加速恶性变细胞周期进程促进细胞增殖，进而引发细胞恶性变。补充精氨酸能够逆转IFN-γ诱导的精氨酸耗竭，并抑制精氨酸耗竭导致的BMEC对细菌的易感性、泌乳品质下降、细胞恶性变和小鼠肿瘤生长。综上，本课题阐明了IFN-γ对奶牛乳腺上皮细胞精氨酸代谢调控机制，为奶牛精氨酸异常代谢相关病症的防治提供了新思路。