Purpose To explore the optimal culture concentration of mechano-growth factor (MGF) for the proliferation of rat skeletal satellite cells (SCs) in vitro. Methods The purified skeletal muscle satellite cells were obtained from a healthy male SD rat. In this study, we adopted an improved two-step enzymatic digestion method combined with differential attachment technique. At the third passage, skeletal satellite cells were treated with MGF concentration of 15, 25, 50, 100, 200 ng/ml.Cells in control group received 100μl growth medium, and in negative control group treated with 100 μl Dulbecco modified Eagle medium (DMEM) . After 24 hours of culture, cholecystokinin-octopeptide (CCK-8) was added. The most appropriate concentration of MGF for promoting cell proliferation was detected by CCK-8 assay. BCA assay and PI assay were utilized to detect the total protein and the apoptosis respectively. And immunocytochemical method was used to identify skeletal muscle satellite cells.Results ( 1 ) As compared with the control group, the MGF concentrations of 15, 25, 50, 100 ng/ml significantly promoted proliferation of SCs (P ＜ 0.01 ) , among which 25 ng/ml had the best effect. (2) As compared with control group, left shift of the SCs growth curve in experimental group appeared, and the doubling time and the flat-top period shortened. (3) As compared with the control group: 25 ng/ml of MGF had a significant effect on SCs at 48 hours (P ＜ 0.05) and at 72 hours (P ＜0.01 ) ; the total protein of SCs increased (P ＜ 0.05 ) and the apoptosis index of skeletal muscle SCs decreased at 72 h and 96 h (P ＜ 0.05) . Conclusion ( 1 ) MGF promotes the proliferation of rat skeletal muscle SCs in vitro and its optimal concentration is 25 ng/ml. (2) 25 ng/ml of MGF also promotes skeletal muscle satellite cells entering to the plateau in advance and thus shortens the growth cycle. (3) 25 ng/ml of MGF inhibits the apoptosis of skeletal muscle satellite cells.%目的:研究机械生长因子(MGF)促离体培养的骨骼肌卫星细胞增殖的最佳浓度,并观察该浓度对细胞生长曲线、总蛋白及凋亡情况的影响.方法:选用雄性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.取第3代骨骼肌卫星细胞,分别采用终浓度为15、25、50、100、200 ng/ml的MGF进行干预,作为实验组;另以单纯加入100 μl生长培养基作为对照组,以加入100μl DMEM作为阴性对照组.同步化后,于培养24 h后加入CCK-8溶液.采用CCK-8比色法检测不同浓度MGF下细胞的增殖情况并筛选出最佳浓度后,绘制生长曲线,同时运用BCA法及Hoechst 33342、PI双染法测定最佳增殖浓度的MGF对卫星细胞蛋白合成及细胞凋亡的影响.免疫细胞化学法鉴定骨骼肌卫星细胞.结果:①终浓度为15、25、50、100 ng/ml的MGF均有促进骨骼肌卫星细胞增殖的作用(P＜0.01),其中25 ng/ml MGF促增殖作用最佳.②与对照组相比,MGF组细胞生长曲线左移,倍增时间、平台期均缩短.③与对照组相比,25 ng/ml MGF作用48 h即对骨骼肌卫星细胞有明显的增殖效果(P＜0.05);与对照组相比,25 ng/ml MGF作用骨骼肌卫星细胞48 h和72 h时,其总蛋白含量显著增加(P＜0.05);与对照组相比,25 ng/ml MGF作用72h、96h,骨骼肌卫星细胞的平均凋亡指数显著减小(P＜0.05).结论:①机械生长因子可促进体外培养的大鼠骨骼肌卫星细胞增殖,最佳浓度为25 ng/ml;②25 ng/ml MGF可以使骨骼肌卫星细胞提前进入平台期,缩短生长周期;③25ng/ml MGF可以抑制骨骼肌卫星细胞的凋亡.
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