马肉成熟过程中肌细胞自噬及其与凋亡的交互作用研究

Tenderness is regarded as the most important criterion for meat quality. The essential role of apoptosis in meat aging and tenderness has been proved. But it is not clear that the mechanism of autophagy, which as another major cell death pattern and is important in the relationship between autophagy and apoptosis, in meat aging is still not clear. To solve this issue, autophagy inducers are used to treat skeletal muscle of horse during the postmortem aging process to enhance the occurrence of autophagy. Then the role of autophagy effect factors in the postmortem aging process will be studied, such as Beclin-1, LC3, and autophagy related signaling pathway mTOR, PI3K/Akt, and so on. The interaction between autophagy and apoptosis during the postmortem aging was investigated by using the inhibitors of autophagy and apoptosis. The relationship between autophagy and apoptosis will be determined that the two are mutually promoting or antagonistic, or at the same time. Then, the key regulation factors of autophagy and apoptosis signal pathway will be analyzed. It will make sure whether these factors play a major role on myofibrillar disruption during horse postmortem aging, and ultimately determine its role in improve the tenderness of meat. The results of this study will complete the existing mechanism of meat aging and offer a new insight and theoretical guidance for improving meat quality.

嫩度是肉类品质最重要的指标之一，细胞凋亡在肉类成熟嫩化中的重要作用已被证实。但是细胞自噬这一与细胞凋亡有着重要相互关系的肌细胞死亡方式，在肉类成熟中的作用机制尚不清楚。本项目以马肉为研究对象，利用自噬诱导剂处理宰后成熟过程中的马肉，加剧肌细胞自噬的发生。研究自噬效应因子Beclin-1、LC3以及自噬相关信号通路mTOR、PI3K/Akt等在宰后成熟中的作用。进一步分别利用自噬和凋亡的抑制剂研究在宰后马肉成熟过程中的细胞自噬与凋亡的交互关系，确定两者是相互促进还是拮抗，或者是独立发生。分析宰后肌细胞自噬与凋亡通路中的关键调节因子对肌原纤维降解的影响，最终确定其在肉嫩度改善中的作用。项目成果将完善现有的肉类成熟理论，为改善肉的嫩度提供新的思路和理论指导。

项目揭示了宰后缺氧缺血及雷帕霉素和3-甲基腺嘌呤处理对成肌细胞系C2C12的影响关系及宰后肉成熟过程中细胞自噬对细胞凋亡的影响关系；明确了了低压电刺激对宰后马肉成熟过程中品质的影响及超声波结合外源酶等处理对马肉饼品质的影响。研究结果如下：1. 利用免疫印迹法检测Beclin-1、LC3及自噬相关信号通路mTOR、P62的变化，发现缺氧缺血条件下C2C12细胞系存在自噬。该条件下C2C12细胞系除自噬还存在凋亡。发现雷帕霉素浓度使用1000nM，处理后激活自噬，抑制凋亡，3-MA浓度使用10mM，处理后抑制自噬及凋亡。2. 研究雷帕霉素和3-MA处理对死亡受体通路及细胞线粒体凋亡通路的影响，发现雷帕霉素处理后，Fas和Fas-L表达水平升高，骨骼肌细胞中caspase-8表达降低，3-MA处理则相反，两种处理间差异不显著。雷帕霉素处理使Bax的蛋白表达增加但不明显，细胞色素C的表达明显增加，3-MA处理则相反。经雷帕霉素处理后肉样caspase-9酶原片段强度显著降低，说明自噬的激活促进了caspase-9酶原的激活，通过线粒体途径促进细胞凋亡，经3-MA处理后肉样的caspase-9酶原片段强度显著升高，说明自噬的抑制减弱了细胞凋亡。3. 通过低压电刺激诱导马肉宰后成熟过程中肌细胞的自噬和凋亡过程，研究发现低压电刺激处理提升了马肉样本僵化、解僵及熟化的速度，加速了马肉骨架大分子蛋白降解，破坏了马肉内部组织完整性，改善了马肉嫩度。4. 超声波辅助复合酶处理的马肉饼水溶性总氮含量、肌原纤维小片化指数、可溶性蛋白浓度、胶原蛋白溶解性显著增加，肌原纤维蛋白含量显著下降，大分子蛋白水解，微观结构呈现均匀细腻、孔洞较少的三维立体网络结构。研究成果为肉类成熟理论提供了一定的科学数据，对肉质嫩度改善具有理论意义及应用价值。