机械生长因子促骨骼肌卫星细胞增殖作用途径的初步研究

Research on the Effect of Mechano Growth Factor on Signaling Pathways and Proliferation of Skeletal Muscle Satellite Cells

目的:研究机械生长因子促进骨骼肌卫星细胞增殖的量效关系及可能的作用途径。方法:选用4周龄雄性SD大鼠1只,无菌条件下取后肢肌肉,采用改进的两步酶消化法结合差速贴壁技术,分离及纯化骨骼肌卫星细胞。免疫细胞化学法鉴定骨骼肌卫星细胞。取第3代骨骼肌卫星细胞,随机分为对照组、DMEM对照组、MGF组(M组)、LY+MGF组(LM组)、PD+MGF组(PM组)和LY+PD+MGF组(LPM组),同步化后,相应组细胞换用含有PI3K特异性抑制剂LY294002(30μM)及MEK特异性抑制剂PD98059(50μM)的生长培养基预阻断12 h后,再用含有MGF(25 ng/ml)的生长培养基继续培养24 h后,用CCK-8比色法、BCA比色法观察不同抑制剂干预下SC的增殖及总蛋白含量。结果:1、PM组和LPM组OD值均显著低于M组(P<0.01),而LM组OD值与M组无显著差异。2、LM组、PM组、LPM组SC总蛋白含量均显著低于M组。结论:MEK/Erk途径在MGF对卫星细胞促增殖方面有较重要作用。

Objective To explore effects of various concentrations of mechano growth factor（MGF）on the signaling pathways and proliferation in rat skeletal satellite cell（SC）in vitro.Methods The primary passage SC was derived from gastrocnemius and soleus of the male SD rat with modified two-step enzymatic digestion method combined with differential attachment technique,and identified by the immunocytochemical method.The 3rd passage SC were divided randomly into control group C（treated with growth medium）,DMEM control group（treated with DMEM）,group M（treated with 25 ng/ml MGF＋ growth medium）,group LM（treated with 30 μM LY294002 ＋ 25 ng/ml MGF ＋ growth medium）,group PM（treated with 50 μM PD98059 ＋ 25 ng/ml MGF ＋ growth medium）,and group LPM（treated with 30μM LY294002 ＋ 50 μM PD98059 ＋ 25 ng/ml MGF ＋ growth medium）.The SC proliferation rate and total protein were measured with CCK-8 assay and BCA assay separately.Results（1）The OD values of groups PD and LPM were significantly lower than group M（P 0.01）.while there was no difference in OD values between groups LM and M.（2）The total protein contents of groups LM,PM,LPM were significantly lower than group M（P 0.01）.Conclusion The signaling pathway of MEK/Erk plays an important role in the proliferation of SC under the action of MGF.