去乙酰化酶Sirt3抗氧化应激作用机制及其与PSE肉形成的关系

With the improvement of life standards, higher requirements have been raised to meat quality in meat products consumption. Stress has great impacts on meat quality, which is the main cause of PSE meat. The various stresses will eventually cause cellular oxidative stress. In the process of oxidative stress, the content of active oxygen species (ROS) will increase, which results in a toxic effect on cells. However, there is a series of cellar mechanisms of oxidative stress resistance to response to the damage caused by oxidative stress. Foreign pig breeds and Chinese local pig breeds have different resistance to oxidative stress, indicating that different breeds have different molecular mechanisms of resistance to oxidative stress. Sirt3 functions as an important oxidative stress resistance protein. In our previous study, we found that the expression of Sirt3 differ significantly in skeletal muscle of Tongcheng pig and Landrace pig. Based on this, we plan to analyze Sirt3 substrates' expression pattern in Tongcheng pig and Landrace pig, construct oxidative stress model using the skeletal muscle primary cell of each breed, and prepare Sirt3 transgenic mice, in order to elucidate the Sitr3 differential expression and its molecular mechanisms in oxidative stress resistance, as well as its effects on the meat characteristics at molecular, cellular and individual levels.

随着生活水平的提高，人们在肉产品消费过程中，对肉的品质有着更高的要求。应激对于肌肉的品质具有重要的影响，是导致屠宰后PSE等劣质猪肉产生的重要原因。机体受到的各种应激最终都会引起细胞的氧化应激。在氧化应激的过程中，活性氧自由基ROS的含量会升高，对细胞具有毒害作用。在细胞内还存在一系列的抗氧化应激机制，应对氧化应激对细胞造成的损害。进口猪种与中国地方猪种拥有不同的抗应激能力，预示着不同猪种在抗氧化应激方面具有不同的分子机制。本课题组前期研究结果表明，抗氧化应激的重要蛋白Sirt3在通城猪及长白猪的骨骼肌中的表达具有显著的差异性。本项目通过具体分析通城猪及长白猪中Sirt3底物蛋白的表达特征，构建不同猪种原代骨骼肌细胞氧化应激模型，制备Sirt3过表达小鼠模型，从分子、细胞及个体水平，揭示不同猪种间，Sirt3差异性表达与抗氧化应激的联系，阐明Sirt3影响肉质性状的具体机制。

品种、环境、疾病等因素都可以影响猪的应激，在应激状态下，会引起细胞氧化应激，导致猪肉品质的下降。为了了解影响猪肉品质的相关基因分子调控机制，本项目利用高通量测序技术鉴定了不同猪种miRNA表达模式并获得差异表达miRNA，通过靶基因预测，获得miRNA及氧化应激相关基因网络调控图；构建骨骼肌细胞氧化应激模型，通过对Sirt3的干扰，检测氧化应激状态下总蛋白的乙酰化水平及H2O2的富集情况，进而阐明骨骼肌细胞氧化应激适应性调节的潜在机制；选取猪PSE及正常肉，研究不同肉质内源性Sirt3的表达，总蛋白的乙酰化水平，以及H2O2的富集情况，解析Sirt3对PSE肉形成的影响。研究结果表明，不同猪种具有不同的miRNA表达特征，表明miRNA对氧化应激相关基因的调控在不同猪种中具有不同的模式；在骨骼肌细胞氧化应激模型中，干扰Sirt3的表达，可使肌肉细胞总蛋白的乙酰化水平升高，H2O2的含量增加，细胞的抗氧化应激的能力下降；与正常肉相比，PSE肉Sirt3表达下降，总蛋白的乙酰化水平较高，H2O2大量富集，表明PSE肉中Sirt3的去乙酰化酶活性可能较低，使得抗氧化应激酶系统相关蛋白的活性受高乙酰化抑制，H2O2等活性氧自由基大量富集，导致肉质品质下降。本项目分别从分子、细胞及个体水平研究了氧化应激相关基因的表观调控、以及Sirt3对细胞氧化应激及猪肉质性状的影响。这些研究内容对于今后的肉质性状决定机制、相关基因挖掘、肉质改善以及分子育种等具有一定的指导意义。