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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 study the dose-response relationship and possible pathways of mechanical growth factor to promote skeletal muscle satellite cell proliferation. METHODS: One male Sprague-Dawley rat aged 4 weeks was selected. Muscle of hindlimb was taken under aseptic conditions. Skeletal muscle satellite cells were isolated and purified by a modified two-step enzyme digestion combined with differential adherent technique. Identification of skeletal muscle satellite cells by immunocytochemistry. The third generation skeletal muscle satellite cells were randomly divided into control group, DMEM control group, MGF group (M group), LY + MGF group (LM group), PD + MGF group (PM group) and LY + PD + MGF group (LPM group). After synchronization, the corresponding groups of cells were pre-blocked with growth medium containing PI3K-specific inhibitor LY294002 (30μM) and MEK-specific inhibitor PD98059 (50μM) for 12 h, 25 ng / ml) growth medium for 24 h, CCK-8 colorimetry and BCA colorimetry were used to observe the proliferation and total protein content of SC under different inhibitors. OD value of PM group and LPM group was significantly lower than that of M group (P <0.01), while OD value of LM group was not significantly different from that of M group. The total protein content of SC in LM group, PM group and LPM group was significantly lower than that in M group. Conclusion: MEK / Erk pathway plays an important role in the proliferation of satellite cells in MGF.