热休克蛋白27对宰后牛肉嫩化的调控机制研究

Tenderness has been considered one of the most important eating quality characteristics of meat. Proteolysis of key myofibrillar proteins by caspase, calpain or other endogenous enzymes with the regulation of various regulatory factors is the largest contributing factor to meat tenderness. The proteomics results demonstrated that heat shock protein 27 (HSP 27) showed a negative correlation with the tenderness of postmortem skeletal muscle and maybe played an important regulation role during the postmortem tenderization process. However, the regulation mechanism of HSP 27 during the process is not fully elucidated. The objective of the study is to investigate the expression of HSP 27 and its subcellular location and translocation during postmortem tenderization process. Meanwhile, the immune-precipitation of HSP 27 with myofibrils, calpain, caspase and apoptotic factors (Bcl-2、Bax and so on) , the structural modification of myofibrils from sHSPs and the changes in susceptibility of modified myofibrils to degradation by the endogenous enzymes in muscles, the influence of HSP 27 on the activity of caspase and calpain and on the expression of apoptotic factors (Bcl-2、Bax) were also examined by applying of SDS-PAGE and western blotting, co-immunoprecipitation, immunohistochemistry, and so on. The results above will contribute to demonstrate the regulation mechanism of HSP 27 associated with tenderness during postmortem tenderization of beef muscle, which is helpful to supply newly theoretical evidence for developing the meat tenderization mechanism.

嫩度是决定肉食用品质的一个重要指标。宰后牛肉嫩化是多因子调控下细胞凋亡酶、钙激活酶等内源酶对肌原纤维蛋白的有限降解。蛋白质组学的研究数据表明热休克蛋白27与嫩度之间存在显著负相关，其在嫩化中可能发挥重要调控作用，但对其调控机理还缺少直接的实验数据支持。本项目旨在综合运用蛋白质印迹、免疫共沉淀、免疫组化等技术，明确热休克蛋白27在牛肉嫩化过程中的亚细胞定位、移位及其表达量变化，重点研究热休克蛋白27与肌原纤维蛋白、细胞凋亡酶和钙激活酶的结合及对其结构修饰，并揭示修饰后肌原纤维蛋白对肌肉内源酶水解敏感性变化以及修饰后钙激活酶或细胞凋亡酶对肌原纤维蛋白的水解活性变化，探究热休克蛋白27对钙激活酶抑制蛋白（calpastatin）和凋亡调节因子（Bcl-2、Bax等）表达量及调节功能的影响。通过以上研究可基本阐明热休克蛋白27对宰后牛肉嫩化的调控机理，从而为完善和发展肉的嫩化机制提供理论依据。

嫩度是决定肉食用品质的一个重要指标，但对宰后嫩化机理仍不十分清楚。目前研究结果表明宰后肌细胞以凋亡的形式死亡，细胞凋亡酶和钙激活酶对肌原纤维蛋白的有限降解是宰后嫩度改善的主要原因。前期研究发现热休克蛋白27与宰后嫩度之间显著相关，本项目主要对其潜在机理进行探究。从离体模型和原位模型两方面，明确了热休克蛋白27对肌原纤维蛋白降解无直接影响，但可与细胞凋亡酶和钙激活酶结合，显著抑制其宰后过程中的表达量、酶活性和其对伴肌球蛋白、伴肌动蛋白、肌间线蛋白等与嫩度相关的肌原纤维蛋白水解能力，间接延缓宰后牛肉嫩化过程。利用免疫定位技术揭示了宰后氧化应激环境下，除热休克蛋白27被逐步被释放，线粒体也被氧化损伤，造成细胞色素c释放，通过线粒体通路激活细胞凋亡酶，两者可能共同通过调控内源酶影响宰后嫩化过程。采用TMT技术对热休克蛋白27抑制前后的肉样进行蛋白质组定量研究，共检测992个差异蛋白，其中与嫩度相关的蛋白597个，在细胞组分分类中主要富集在超分子聚合物、细胞外区域、肌原纤维、收缩纤维、肌节、I带、Z线、A带等结构上，解析了热休克蛋白27可能参与宰后氧化磷酸化钙信号传导途径等关键生化通路。研究结果可为完善和发展肉的宰后嫩化机制提供新的理论依据，为进一步建立以热休克蛋白27为靶向的宰后肌肉嫩化调控技术提供技术支撑。