cys蛋白酶-3对离体培养的骨骼肌蛋白降解的影响

The influence of caspase3 to the cultured skeletal muscle cells' proteolysis

目的:通过体外培养骨骼肌细胞,测定凋亡效应相关蛋白酶cys蛋白酶-3(caspase-3)及其抑制剂对骨骼肌细胞蛋白降解率的影响,探讨骨骼肌细胞凋亡的可能的调控因子。方法:0.5μg、1μg、2μg、4μg、8μg的肌动球蛋白复合体的反应体系中加入1单位的重组Caspase-3,通过Western Blot电泳。体外培养骨骼肌细胞,并刺激其形成肌管,将培养的骨骼肌分为3组,分别为对照组、实验组A加入1单位Caspase-3,实验组B加入50 nM Caspase-3的抑制剂Ac-DEVD-CHO,体系中均加入2mM ATP(三磷酸腺苷)。用氨基酸全谱分析仪测定酪氨酸和3-甲基组氨酸的浓度。结果:0.5μg、1μg、2μg、4μg、8μg的肌动球蛋白复合体和1单位的重组Caspase-3,均可见到14kDa-actin条带,而肌动球蛋白复合体在0.5μg、1μg剂量时条带消失,在2μg、4μg、8μg时条带逐渐增大。与对照组相比,给予caspase-3后骨骼肌细胞总蛋白降解率增加36.9%,肌纤维蛋白降解率增加66.4%,给予DEVD后总蛋白降解率下降17.7%,肌纤维蛋白降解率下降27.7%。结论:caspase 3在体外可以降解肌动球蛋白复合体,而且还能改变骨骼肌蛋白降解率。caspase-3在骨骼肌蛋白降解中发挥了重要作用,参与了骨骼肌细胞的凋亡。

Objective With the help of skeletal muscle cell culture,the influence of apoptosis caspase3 and its inhibitor on the skeletal muscle proteolytic rate were detected.The possible regulatory factors of apoptosis on the skeletal muscle wasting was studied.Methods 1 unit of caspase3 was added into 0.5μg,1μg,2μg,4μg and 8μg actomyosin respectively.The reaction products were determined by Western Blot.The skeletal muscle cells were cultured in vitro and were stimulated into forming myotube.The cultured cells were devided into 3 groups including the control group and the treatment group A and B.1 unit caspase3 was added into the treatment group A.50 nM Ac-DEVD-CHO,one of caspase3 inhibitors,was added into the treatment group B.2mM ATP were added into all of the three groups.The concentrations of tyrosine and 3-methylhistidine were determined by amino acid analyzer.Results 14kDa-actin small fragment were observed in all of the actomyosin and caspase3 reaction systems.While the actomyosin bands were disappear in 0.5μg and 1μg reaction systems and increased gradually in 2μg,4μg and 8μg reaction systems.Compared with the control group,with the help of caspase3 the skeletal muscle proteolytic rate was increased by 36.9% and the muscle fiber proteolytic rate was increased by 66.4%.When DEVD was given,the skeletal muscle proteolytic rate was decreased by 17.7% and the muscle fiber proteolytic rate was decreased by 27.7% compared with the control group.Conclusion Caspase3 could breakdown actomyosin complex and change the skeletal muscle proteolytic rate.Caspase3 plays an important role in skeletal muscle protein degradation and takes part in the apoptosis of skeletal cells.