外源性机械生长因子对大鼠骨骼肌卫星细胞生物学特性的影响

Effect of Exogenous Mechano-Growth Factor on the Biological Characteristics of Skeletal Satellite Cells in Rats

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100μl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;③25 ng/ml MGF可以抑制骨骼肌卫星细胞的凋亡。

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.