Skeletal muscle cell activation by atmospheric plasma exposure and application to tissues

放电等离子体是一种部分电离气体，含有自由基、电子、正负离子和各种激发原子。近年来，许多类型的低温大气压等离子体装置已经被开发出来用于医疗应用。在本研究中，我们研究了低温大气等离子体暴露对C2C12成肌细胞增殖和分化的影响。低温大气压气体等离子体是在氩气中通过放电产生的。每24小时1分钟的血浆暴露可抑制细胞增殖，但未见细胞死亡。染毒后24 h和48 h，染毒组细胞数分别为对照组的42.2%和32.9%。另一方面，成肌细胞分化的标志性蛋白，如肌生成素和肌球蛋白重链的表达不受血浆暴露的影响。暴露后30min，细胞外调节蛋白激酶(ERK)和c-jun氨基末端蛋白激酶(JNK)的磷酸化水平升高。而ERK和JNK的磷酸化水平在暴露后1h和4h明显低于对照组，并在作用后2 4h恢复到对照水平，流式细胞仪分析显示，暴露后8h，细胞处于G2/M期的百分率明显增加。研究发现，在血浆暴露的细胞中，调节G2/M检查点的cdc2/Cyclin B1下调。P21WAF-1/Cip-1控制细胞进入G2/M相变检查点，p21WAF-1/Cip-1在血浆暴露细胞中的表达也升高。综上所述，低温大气等离子体暴露通过G2/M期阻滞抑制了C2C12成肌细胞的增殖。ERK和JNK活性的下调以及细胞周期检查点蛋白的调节可能参与了等离子体暴露诱导的细胞周期停滞。

Plasma in electrical discharge is a partially ionized gas, containing radicals, electrons, positive and negative ions, and various excited atoms. Recently, many types of low-temperature atmospheric plasma devices have been developed for medical applications. In the present study, we investigate the effect of low-temperature atmospheric plasma exposure on proliferation and differentiation of C2C12 myoblast. The low-temperature atmospheric pressure gas-plasma was generated through an electrical discharge in an argon gas. One min of plasma exposure every 24 h inhibited the cell proliferation without cell death. Cell numbers in plasma exposure groups were 42.2% and 32.9% when compared to the control groups at 24 h and 48 h after the plasma exposure, respectively. On the other hand, the marker proteins for the myoblast differentiation, such as myogenin and myosin heavy chain expressions were not affected by plasma exposure. Plasma exposure increased the phosphorylation of extracellular-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) at 30 min after the exposure. However, phosphorylation of ERK and JNK was markedly decreased less than control levels at 1 and 4 h after the plasma exposure, respectively, and returned to the control levels at 24 h. By flow cytometric analysis, it revealed that plasma exposure increased the % of the cell in the G2/M phase at 8 h after the exposure. It was found that down-regulation of cdc2/cyclin B1, which regulates G2/M checkpoint in plasma-exposed cells. The expression of p21 Waf-1/Cip-1, which was known to control the entry of cells at the G2/M phase transition checkpoint was also elevated in plasma-exposed cells. In conclusion, low-temperature atmospheric plasma exposure retarded proliferation of C2C12 myoblast by G2/M arrest. Down-regulation of ERK and JNK activity and modulation of cell cycle checkpoint proteins may be involved in plasma exposure-induced cell cycle arrest.