奶牛泌乳和乳腺退化的5-HT调控机制研究

Serotonin (5-HT) has been demonstrated to be an important local regulator of lactational homeostasis and involution. Whereas, the mechanisms employed by 5-HT during regulation of lactation and involution in the bovine mammary gland are poorly understood.The bovine mammary gland expresses multiple functional isoforms of serotonin receptors (HTR1B, 2A, 2B, 4, and 7), and expression of milk protein levels were stimulated by blocking HTR1B and HTR2A with the selective antagonists. In this project, we will use primarily cultured bovine mammary epithelial cells (MEC),to investigate the intracellular signal pathway mediating the serotonin receptors' (HTR1B and HTR2A) action on regulating expression of milk protein; test the effect of 5-HT and selective 5-HT receptor antagonists on proliferation and apoptosis of MEC, and explore which intracellular pathways are involved in the transduction of the serotoninergic intracellular signal; and then determine whether 5-HT induce mammary gland involution indirectly through changing expression level of the factors that regulate proliferation and apoptosis of MEC,such as IGF-I, Fas-l,EGFs and TGF-beta. Addressing the serotonin mechanisms associated with lactation and involution, may help us to unveil general principles of mammary gland biology, promote the development of lactation physiology, and provide theoretical base for improvement of milk production and quality.

5-HT被认为是一种重要的泌乳稳态和退化局部调控因子。但是，对于其调控泌乳及乳腺退化的机制，目前知之甚少。奶牛乳腺表达多种亚型的5-HT受体（HTR1B, 2A, 2B, 4, 和7），选择性阻断HTR1B和HTR2A引起乳蛋白表达量升高。本项目将利用原代培养的奶牛乳腺上皮细胞（Mammary epithelial cells, MEC），探寻HTR1B和HTR2A调控乳蛋白的表达的信号通路；检测5-HT及其各亚型受体选择性拮抗剂对奶牛MEC增殖与凋亡的影响，并探寻其作用机制；确定5-HT是否通过改变IGF-I、 Fas-l、EGFs、TGF-beta?等细胞增殖凋亡调控因子的表达水平间接诱导乳腺退化，以系统揭示5-HT调控奶牛泌乳和乳腺退化的机理。通过探明5-HT的作用机理，有助于进一步揭示乳腺生物学的基本规律，推动泌乳生理学的发展，为提高奶牛乳产量及乳品质提供理论依据。

5-HT被认为是一种重要的泌乳稳态局部调控因子，以旁分泌或自分泌方式调控乳蛋白的合成和乳腺退化，但其机制尚不明确。本研究利用MAC-T细胞系——一种确立的奶牛乳腺上皮细胞系，研究5-HT对细胞增殖凋亡的影响及其作用机制，并探寻5-HT调控乳蛋白合成的信号通路。利用RT-PCR技术，我们在奶牛乳腺组织和MAC-T细胞中检测到5-HT合成限速酶TPH1，以及1B、2A、2B、4、7、5A、3A、1F、1D、2C、1E、1A等多种亚型5-HT受体mRNA的表达。用不同浓度5-HT处理MAC-T细胞12h，24h，48h，72h，发现5-HT通过改变Bax/Bcl-2的值双向调控MAC-T细胞增殖凋亡，即短时间5-HT处理，促进MAC-T细胞的增殖，抑制凋亡；而长时间5-HT处理抑制MAC-T细胞的增殖，促进凋亡。5-HT对MAC-T细胞增殖的促进作用是通过1B、4亚型受体激活mTOR-eEF2信号通路来实现的，而对酪蛋白表达的抑制作用是通过JAK2-STAT5信号通路来实现的。本研究结果为阐明5-HT调控奶牛泌乳稳态机理提供了新的实验依据，有助于进一步探讨和揭示泌乳周期中奶牛乳腺组织细胞更新机制，在人为调控细胞凋亡、加速乳腺退化、预防和治疗乳腺疾病以及提高乳产量和乳质量等方面均将具有重要的学术价值。