血小板源生长因子BB可促进骨骼肌成肌细胞增殖、分化与迁移

Platelet-derived growth factor-BB promotes proliferation,differentiation and migration of skeletal muscle myoblast

背景:骨骼肌损伤后,骨骼肌成肌细胞分化融合形成多核肌管和肌纤维完成肌损伤修复,但该过程修复缓慢且不完全。血小板源生长因子BB可以刺激多种组织细胞增殖、分化及迁移,在各种损伤后组织修复过程中发挥了重要作用。目的:探讨不同浓度血小板源生长因子BB对骨骼肌成肌细胞增殖、分化及迁移的影响及作用机制。方法:培养小鼠骨骼肌成肌细胞(C2C12细胞),分别给予血小板源生长因子BB 0,5,10,20,40μg/L及血小板源生长因子受体抑制剂伊马替尼处理。采用免疫细胞化学法及Western-blot法检测C2C12细胞中血小板源生长因子受体表达情况;分别培养1,2,3,4,5 d后,采用CCK8法检测细胞增殖情况;给予诱导分化培养4 d,采用光学显微镜观察各组肌管的形成情况,通过免疫荧光化学法和Western-blot法观察各组肌管中肌球蛋白重链及MyoG基因的表达情况;培养48 h后,采用Transwell法检测细胞迁移情况。结果与结论:①免疫荧光化学及Western-blot均提示C2C12细胞中可检测到血小板源生长因子受体表达,半定量后统计学分析显示不同血小板源生长因子BB质量浓度组间血小板源生长因子受体表达量差异无显著性意义(P>0.05);②CCK8检测结果显示,与对照组(血小板源生长因子BB 0μg/L组)比较,不同质量浓度血小板源生长因子BB组C2C12细胞增殖无明显改变;③免疫荧光化学法检测结果显示,与对照组相比,血小板源生长因子BB处理组肌球蛋白重链阳性细胞数增多,计数平均每个显微镜视野下肌管形成数目提示血小板源生长因子BB质量浓度为40μg/L时成熟肌管形成数目最多,达(27.00±0.76)个/视野,且该组中MyoG表达数量增多最为明显,与对照组相比差异有显著性意义(P<0.05);④Transwell结果显示,与对照组相比较,不同质量浓度血小板源生长因子BB组C2C12细胞的迁移数量均增加,其中以40μg/L组迁移数目最高达144.00±13.03(P<0.05);⑤提示血小板源生长因子BB能够促进C2C12细胞迁移、分化及其肌管形成,且促分化机制与其增强与血小板源生长因子受体结合、从而提高分化相关基因MyoG的表达有关。

BACKGROUND:Skeletal muscle myoblasts differentiate and fuse to form polynuclear muscle tubes and muscle fibers to complete the repair of muscle injury when skeletal muscles were injured.However,the repair process is slow and incomplete.Platelet-derived growth factor-BB can stimulate the proliferation,differentiation and migration of a variety of tissue cells,and plays an important role in the process of tissue repair after various injuries.OBJECTIVE:To explore the effects of different concentrations of platelet-derived growth factor-BB on proliferation,differentiation and migration of skeletal muscle myoblast C2C12 cells and the action mechanism.METHODS:The mouse skeletal muscle myoblast C2C12 cells were cultured with platelet-derived growth factor-BB 0,5,10,20 and 40μg/L and platelet-derived growth factor receptor inhibitor imatinib.The expression of platelet-derived growth factor receptor in C2C12 cells was detected by immunocytochemistry and western blot assay.After 1,2,3,4,and 5 days of culture,CCK8 method was used to detect cell proliferation.After 4 days of induction and differentiation culture,the formation of myotubes in each group was observed by light microscope.The expression of myosin heavy chain and MyoG Gene was observed by immunofluorescence and western blot assay.After 48 hours of culture,Transwell method was used to detect cell migration.RESULTS AND CONCLUSION:(1)Immunofluorescence chemistry and western blot assay indicated that platelet-derived growth factor receptor expression could be detected in C2C12 cells,and semi-quantitative statistical analysis showed no significant difference in platelet-derived growth factor receptor expression between groups with different platelet-derived growth factor-BB concentrations(P>0.05).(2)CCK8 assay demonstrated that the proliferation of C2C12 cells showed no significant change in platelet-derived growth factor-BB groups compared with the control group(platelet-derived growth factor-BB 0μg/L group).(3)Immunofluorescence chemistry showed that compared with control group,number of myosin heavy chain positive cells increased in platelet-derived growth factor-BB groups;40μg/L platelet-derived growth factor-BB concentration got the highest;the mature muscle tubes up to(27.00±0.76)per field of vision,and MyoG expression populations was most obviously compared with the control group(P<0.05).(4)Transwell results showed that compared with the control group,the migration number of C2C12 cells in the platelet-derived growth factor-BB group increased,and the migration number in the 40μg/L group was up to 144.00±13.03(P<0.05).(5)It is concluded that platelet-derived growth factor-BB promoted the migration,differentiation and myotube formation of C2C12 cells,and the pro-differentiation mechanism is related to its enhanced binding with platelet-derived growth factor receptor,so as to improve the expression of differentiation related gene MyoG.