热应激与抑制素在调控猪颗粒细胞功能中的互作及其分子机理研究

High temperature induced heat stress in summer is one of the most important factors that impact the animal reproduction performance. In our in vitro studies, we found that heat stress could directly suppress the secretion of E2 by granulosa cell, and immunization of Iinhibin could retrieve and increases the secretion, but we know nothing about the molecular mechanisms. And we hypothesized that heat stress induced HSPs suppressed the phosphorylation and nuclear –translocation of Smads, and inhibit the functions of granulosa cell. But when treated with anti-Inhibin antibody, with the dual functions, HSPs would be over expressed and blocked, and its suppressions to Smads were relieved, so the E2 secretion increased. To confirm this hypothesis, studies in this project were mainly started in heat stress and anti-Inhibin antibody induced HSPs expression, Smads phosphorylation and cellular localization. In this project, signaling pathways were blocked and enhanced with small molecular inhibitors and over expression respectively, and then, genes associated with hormones synthesis, protein translation, phosphorylation and cellular localization were assayed. According to the results of RNA-seq, cell cycle, proliferation and apoptosis of the cells treated with heat stress and antibody, the molecular mechanisms of heat stress suppress the functions of cells but Inhibin immunization could retrieve and increase would be discovered finally, and hope that this project could provide theoretical foundations for the practical utilization of Inhibin immunization in production.

夏季高温热应激是影响动物繁殖性能的重要因素之一。我们体外研究发现热应激可抑制颗粒细胞分泌E2，而免疫抑制素可增强其分泌功能，但对与其分子机理尚不清楚。对此我们提出以下假设：热应激通过其诱导的HSPs抑制Smads的磷酸化和核转移而影响细胞功能，但在抗抑制素抗体和热应激的双重作用下，导致HSPs过表达而造成表达抑制，缓解对Smads的抑制作用，增强细胞分泌E2的功能。为证实这一假设，本研究主要从热应激和抗抑制素抗体处理细胞后HSPs的表达、Smads的磷酸化及细胞定位等方面展开。在研究中分别采用小分子抑制剂阻断其信号传导和过表达增强信号传导，然后对细胞中与激素合成相关基因和蛋白的表达变化、磷酸化和细胞定位进行检测。结合转录组测序及热应激和抗体处理对细胞周期、增殖和凋亡等方面的结果，最终揭示热应激影响颗粒细胞功能和免疫抑制素增强热应激细胞功能的分子机理，为抑制素免疫在生产中的应用提供理论依据。

LPS和热应激是影响动物繁殖性能的两个重要的环境因素。目前已有研究证明，LPS和热应激可显著抑制动物的内分泌。卵泡颗粒细胞与卵母细胞关系密切，在动物繁殖活动中发挥重要作用。然而LPS和热应激对颗粒细胞的直接影响及分子机理仍缺乏深入了解。因此，本研究旨在探讨LPS和热应激对颗粒细胞功能的影响和分子机理进行初步研究，并进一步探索抗抑制素抗体对改善颗粒细胞功能的作用机制。本研究采用离体培养的猪颗粒细胞为模型，分别采用LPS, 热应激（41℃）和抗抑制素抗体处理，对细胞内与激素分泌相关基因的表达进行检测，并采用阻断和激活相关信号通路的方式，进一步探索其作用的分子机理。结果发现热应激和LPS可显著抑制芳香化酶P450arom和FSHR基因的表达，同时上调HSP70的表达，免疫荧光染色和Western blot的结果已经证明，上调的HSP70可抑制Smad2/3的磷酸化和核转移，并抑制雌激素的分泌。为模拟HSP70升高的状态，采用HSP70激活剂的方式。通过激活剂处理后，HSP70显著上调，Smad2/3的磷酸化和核转移，雌激素的分泌均受到显著抑制。发现LPS能够促进体外培养的猪颗粒细胞增殖、抑制细胞凋亡、增强细胞活力。表现为细胞形态良好、细胞增殖、活细胞比率增加、凋亡细胞比率减少，Phospho-ERK、Phospho-JNK、Phospho-p38 MAPK蛋白表达量上调，与促进细胞增殖和细胞周期有关的基因FN1、IGF2、IGFBP2、Cyclin D1、Cyclin D2相对表达量上调，抑制细胞周期的基因P27kip相对表达量下调。而采用抗抑制素抗体处理后，可显著上调雌激素合成酶类基因的，促进了信号传导分子Smad的磷酸化及PKA表达的上调加强了信号通路。转录元件FOXL2、CEBP的上调促进了FSHR和TGF-beta家族的表达。本研究的进行进一步完善和发展动物繁殖活动的旁分泌和自分泌调控机制，尤其是热应激导致繁殖障碍的细胞及分子调控机理，阐明了HSP70在抑制颗粒细胞功能中的作用机制，同时为抑制素免疫在实际生产上的推广应用提供理论依据。