香烟提取物对大鼠骨骼肌细胞肌肉生长抑制素表达的影响研究

Influence of Cigarettes Smoke Extract on the Expression of MSTN in SD Rat Skeletal Muscle Cells

目的探讨香烟提取物（CSE）对骨骼肌细胞肌肉生长抑制素（MSTN）表达的影响。方法取新生3-5 d的SD大鼠的骨骼肌组织,用含150 ml/L胎牛血清的高糖DMEM培养基在37℃、5%CO2培养箱内进行体外原代细胞培养和传代。采用免疫荧光鉴定检测骨骼肌细胞。选择恰当的CSE浓度（2.5%、5.0%、10.0%）刺激48 h,分为2.5%组、5.0%组、10.0%组和对照组（不予CSE刺激）,分别采用实时定量PCR检测MSTN mRNA的表达水平与Western blotting检测MSTN蛋白的表达水平。结果骨骼肌细胞培养成活率高,体外生长、增殖良好。实时定量PCR检测结果显示,4组MSTN mRNA相对表达水平比较,差异有统计学意义（P〈0.05）;其中,5.0%组、10.0%组MSTN mRNA相对表达水平高于对照组和2.5%组（P〈0.05）。Western blotting检测结果显示,4组MSTN蛋白表达水平比较,差异有统计学意义（P〈0.05）;其中,5.0%组、10.0%组MSTN蛋白表达水平高于对照组和2.5%组（P〈0.05）。结论骨骼肌细胞模型可对CSE刺激产生有效反应,为吸烟引起骨骼肌抑制的机制研究奠定了细胞学基础。

Objective To investigate the influence of cigarettes smoke extract（ CSE） on the expression of MSTN in skeletal muscle cells. Methods The skeletal muscle tissues from newborn SD rat within 3-5 days old, were cultured and subcultured in high glucose DMEM with 150 ml / L fetal bovine serum in a incubator for proliferation in vitro. The skeletal muscle cells were detected by immunofluorescence technology. The skeletal muscle cells were treated with different levels of CSE（ 2. 5%,5. 0%,10. 0%） for 48 h,and according to CSE concentration,skeletal muscle cells were divided into 2. 5% group,5. 0% group,10. 0% group,and control group（ cells were not treated with CSE） respectively. The expression of MSTN mRNA was detected by RT-PCR,and the expression of MSTN proteins was detected by Western blotting technology. Results The survival rate of skeletal muscle cells was high in vitro,and the growth and proliferation status was good. According to RT-PCR results,there were significant differences in relative expression level of MSTN mRNA among four groups（ P〈0. 05）. The relative expression level of MSTN mRNA in 5. 0% group and in 10. 0% group was significantly higher than that in control group and in 2. 5% group, respectively（ P〈0. 05）. According to Western blotting results, there were significant differences in expression level of MSTN proteins among four groups（ P〈0. 05）. The expression level of MSTN proteins in 5. 0% group and in10. 0% group was significantly higher than that in control group and in 2. 5% group,respectively（ P〈0. 05）. Conclusion Skeletal muscle cell model can effectively respond to CSE stimulation,which provides cytological basis for mechanism research on cigarettes smoke-induced skeletal muscle inhibition.