外泌体介导氨基酸感受器GCN2抑制奶牛乳腺上皮细胞炎症反应的分子机制研究

乳腺炎严重危害奶业健康发展。目前抗生素仍然是其防治的主要手段，但细菌耐药性和药物残留等问题日趋严重。因此，如何安全、有效的防治乳腺炎成为了亟待解决的科学问题。GCN2是氨基酸感受器。申请人首次发现奶牛乳腺组织有GCN2存在，并证实它参与乳腺上皮细胞（MECs）泌乳与细胞增殖调控。但是，GCN2与MECs炎症反应的关系，国内外尚无报道。基于国内外研究进展和前期工作，我们提出在MECs炎症反应过程中通过激活GCN2抑制外泌体传递的促炎信号，进而缓解MECs炎症反应的假设。为证实这一假设，本项目拟采用PCR-Arrays、免疫印迹、RNA沉默、EMSA等技术，在体外MECs炎症模型上系统研究GCN2调节促炎细胞因子和促炎酶类表达以及外泌体释放规律，阐明外泌体触发炎症反应的关键信号通路，并在小鼠乳腺炎模型上一一验证，揭示GCN2在乳腺炎发生发展中的作用及其分子机制，以期为乳腺炎的防治提供新靶点。

Mastitis seriously endangers the healthy development of dairy industry. At present, the treatment of mastitis is largely depends on the usage of antibiotics. However, the emergence of drug-resistance strain and antibiotic residue in animal products highlight the need for more safe and effective therapy. GCN2 is an amino acid sensor. Our previous studies found for the first time that GCN2 was expressed in dairy cow mammary gland, and it may exert a regulatory role in lactation and cell proliferation in mammary epithelial cells (MECs). However, there is no literature reported regarding the relationship between GCN2 and LPS-induced inflammation in MECs both at home and abroad. Based on the research progress at home and abroad and our preliminary work, we hypothesis that halofuginone-activated GCN2 inhibits exosome-dependent pro-inflammatory message, and further alleviates the inflammatory response of LPS-induced inflammation in MECs. The PCR-Arrays, immunoblotting, RNA silencing, EMSA and other technologies will be used to explore the regulatory role of GCN2 on the expression of proinflammatory cytokines and proinflammatory enzymes and the release of exosomes at the molecular and cellular level for confirming our hypothesis and for clarifying the key signaling pathways of exosomes triggered inflammatory responses. Finally, the in vitro results will be verified in mice model one by one, revealing the anti-inflammatory effects and the underlying mechanisms of GCN2 in mastitis pathogenesis. Our finding will provide an alternative therapy target of mastitis.