GRα介导钙离子信号调节猪骨骼肌细胞应激凋亡的研究

项目名称： GRα介导钙离子信号调节猪骨骼肌细胞应激凋亡的研究

项目编号： No.31301946

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 农业科学

项目作者： 柴进

作者单位： 华中农业大学

项目金额： 21万元

中文摘要： 近年来猪应激后肉质下降与应激激素糖皮质激素（GC）之间的关系倍受人们关注，但迄今为止，GC含量升高导致肉质劣变的机理尚不清楚。前期研究中，我们发现高浓度的GC可致使骨骼肌细胞凋亡且胞质中钙离子大量聚集。我们推测，升高的GC可能通过GRα调控钙离子通道，导致胞质中钙离子升高而激发依赖钙离子的细胞凋亡信号，引起肌细胞的应激凋亡，致使肌细胞的理化状态变化，进而影响肉质。本项目利用猪原代肌细胞应激模型，采用流式细胞术、定量PCR、免疫印迹、定点突变、凝胶电泳迁移实验、染色质免疫共沉淀、过表达与干扰等手段探讨钙离子信号紊乱对猪肌细胞存活的影响、GC对钙离子信号的调控作用、GRα对钙离子通道基因的调控机制，并在体内试验中验证GC肌细胞凋亡、钙离子信号与肉质下降之间的关系。本课题将进一步挖掘应激状态下肉质劣变的内在机制，为选育抗应激和高品质肉质猪，以及采取合理的措施减少应激造成的危害提供一定的理论依据。

中文关键词： 猪；应激；糖皮质激素；肉质；分子机制

英文摘要： Recently, much attention has been focused on the relationship between poor meat quality and glucocorticoid （GC） caused by stress. However, molecular mechanism under the elevated GC levels led to poor meat quality is not very clear so far. We previously showed that high concentrations of GC led to skeletal muscle cell apoptosis accompanied with high calcium level appeared in the cytoplasm. Then, we infer that the elevated GC may regulate calcium channels through glucocorticoid receptor α (GRα), subsequently leading to increased calcium in the cytoplasm and to further stimulate the calcium-dependent apoptotic signal, which could in turn affect meat quality. On the basis of our previous study, we use pig primary muscle cells to build a cellular stress model and then to study how calcium signaling disorders affect the survival of porcine muscle cells, how GC control the calcium signaling pathway and how GRα regulates calcium channel gene by this cell model using flow cytometry, quantitative PCR, western blot, site-directed mutagenesis, electrophoretic mobility shift assay (EMSA), chromatin coimmunoprecipitation (ChIP), over expression and siRNA method. And we also want to verify the relationship between GC, calcium signal, muscle cell apoptosis and poor meat quality in vivo model. The present study is of significant

英文关键词： porcine；stress；glucocorticoids；meat quality；molecular mechanisms