香烟对小鼠C2C12成肌细胞分化的影响

Effect of cigarette smoking condensate on myogenic differentiation of murine skeletal muscle C2C12 cells

目的:探讨香烟烟雾浓缩物(CSC)对小鼠骨骼肌C2C12成肌细胞分化的影响。方法:小鼠骨骼肌成肌细胞C2C12传代后分为对照组、0.3%CSC孵育组、3%CSC孵育组,细胞在含有马血清的DMEM培养基第6-7d可分化成熟的肌细胞。细胞分别在第1d、第3d在相应组中加入CSC,并在第6d进行各项检测:通过光学显微镜来观察细胞形态的变化,通过酶联免疫检测仪检测细胞内谷胱甘肽(GSH)和肌酸磷酸激酶(CK)的浓度,应用Western印迹检测肌球蛋白重链(MYHC)的蛋白表达,通过凝胶迁移率阻滞实验来检测转录因子核因子-κB(NF-κB)的活性。结果:在细胞形态上0.3%和3%的CSC能够抑制骨骼肌肌管的形成;0.3%和3%CSC处理组细胞中GSH和CK的含量较对照组明显减少,在0.3%和3%的CSC孵育组中MYHC蛋白表达明显减少,0.3%和3%的CSC孵育组中NF-κB的活性较对照组明显增强。结论:CSC一方面加重细胞氧化应激,使细胞的GSH减少,同时导致细胞的炎症反应增加,导致NF-κB的活性增加。二者相互作用引起骨骼肌细胞的分化明显减弱。

AIM：To study the effect of cigarette smoking condensate （CSC） on myogenic differentiation of murine skeletal muscle C2C12 cells. METHODS：Murine skeletal muscle C2C12 cells,which have the ability to differen-tiate into muture muscle cells,were cultured in high glucose-containing DMEM complemented 10% horse serum and were divided into control group,0. 3% CSC group and 3% CSC group. The cells were incubated with 0. 3% or 3% CSC for 1 day or 3 days,and the detection was performed on 6th day. The morphologic changes of the cells were observed under microscope. Intracellular concentrations of creatine kinase （CK） and glutathione （GSH） were measured by spectrophotometric assay. The activation of the transcription factor nuclear factor-κB （NF-κB） in the cells was evaluated by electrophoretic mobility shift assay （EMSA）. The protein expression of myosin heavy chain （MYHC） was detected by Western blotting. RESULTS：CSC at concentrations of 0. 3% and 3% inhibited the formation of myotube in C2C12 cells. C2C12 cells treated with CSC significantly reduced the intracellular levels of GSH and CK as compared to those in control cells. Treatment with CSC also inhibited the activation of NF-κB and the expression of MYHC. CONCLUSION：CSC inhibits the differentiation of C2C12 cells by decreasing the level of antioxidatant GSH and increasing the activity of NF-κB.