cys蛋白酶-3對(duì)離體培養(yǎng)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(yǎng)骨骼肌細(xì)胞，測(cè)定凋亡效應(yīng)相關(guān)蛋白酶cys蛋白酶－3（caspase-3）及其抑制劑對(duì)骨骼肌細(xì)胞蛋白降解率的影響，探討骨骼肌細(xì)胞凋亡的可能的調(diào)控因子。方法：0.5μg、1μg、2μg、4μg、8μg的肌動(dòng)球蛋白復(fù)合體的反應(yīng)體系中加入1單位的重組Caspase-3，通過Western Blot電泳。體外培養(yǎng)骨骼肌細(xì)胞，并刺激其形成肌管，將培養(yǎng)的骨骼肌分為3組，分別為對(duì)照組、實(shí)驗(yàn)組A加入1單位Caspase-3，實(shí)驗(yàn)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(cè)定酪氨酸和3-甲基組氨酸的濃度。結(jié)果：0.5μg、1μg、2μg、4μg、8μg的肌動(dòng)球蛋白復(fù)合體和1單位的重組Caspase-3，均可見到14kDa-actin條帶，而肌動(dòng)球蛋白復(fù)合體在0.5μg、1μg劑量時(shí)條帶消失，在2μg、4μg、8μg時(shí)條帶逐漸增大。與對(duì)照組相比，給予caspase-3后骨骼肌細(xì)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(jié)論：caspase 3在體外可以降解肌動(dòng)球蛋白復(fù)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(fā)揮了重要作用，參與了骨骼肌細(xì)胞的凋亡。

[關(guān)鍵詞]骨骼肌；蛋白代謝；細(xì)胞培養(yǎng)；凋亡

[中圖分類號(hào)]Q591.2[文獻(xiàn)標(biāo)識(shí)碼]A[文章編號(hào)]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