cys蛋白酶-3對離體培養的骨骼肌蛋白降解的影響

吳焱秋 柴家科 張海龍 耿祎楠

[摘要]目的：通過體外培養骨骼肌細胞，測定凋亡效應相關蛋白酶cys蛋白酶－3（caspase-3）及其抑制劑對骨骼肌細胞蛋白降解率的影響，探討骨骼肌細胞凋亡的可能的調控因子。方法：0.5μg、1μg、2μg、4μg、8μg的肌動球蛋白復合體的反應體系中加入1單位的重組Caspase-3，通過Western Blot電泳。體外培養骨骼肌細胞，并刺激其形成肌管，將培養的骨骼肌分為3組，分別為對照組、實驗組A加入1單位Caspase-3，實驗組B加入50 nM Caspase-3的抑制劑Ac-DEVD-CHO，體系中均加入2mM ATP(三磷酸腺苷)。用氨基酸全譜分析儀測定酪氨酸和3-甲基組氨酸的濃度。結果：0.5μg、1μg、2μg、4μg、8μg的肌動球蛋白復合體和1單位的重組Caspase-3，均可見到14kDa-actin條帶，而肌動球蛋白復合體在0.5μg、1μg劑量時條帶消失，在2μg、4μg、8μg時條帶逐漸增大。與對照組相比，給予caspase-3后骨骼肌細胞總蛋白降解率增加36.9%，肌纖維蛋白降解率增加66.4%，給予DEVD后總蛋白降解率下降17.7%，肌纖維蛋白降解率下降27.7%。結論：caspase 3在體外可以降解肌動球蛋白復合體，而且還能改變骨骼肌蛋白降解率。caspase-3在骨骼肌蛋白降解中發揮了重要作用，參與了骨骼肌細胞的凋亡。

[關鍵詞]骨骼肌；蛋白代謝；細胞培養；凋亡

[中圖分類號]Q591.2[文獻標識碼]A[文章編號]1008-6455（2014）07-0541-04

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

WU Yan-qiu,CHAI Jia-ke,ZHANG Hai-long,GENG Yi-nan

(Burns Institute,The First Affiliated Hospital to the General Hospital of PLA,Beijing 100048, China )

Abstract:ObjectiveWith 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.Results14kDa-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.

Key words:skeletal muscle;protein degradation;cell culture;apoptosis