冲击波对成肌细胞增殖分化的影响

关键词：冲击波  成肌细胞  增殖  分化  生物学功能

Keywords:shock wave  myoblast  proliferation  differentiation  biological functions

摘要：

      摘要 目的：探讨体外冲击波对成肌细胞增殖分化的影响，为进一步开展冲击波在体内骨骼肌损伤中的作用研究奠定基础，为临床应用冲击波治疗提供理论依据。 方法：将原代分离培养好的成肌细胞，利用冲击波进行刺激，将实验对象分为空白组、实验组（冲击波刺激）、阳性对照组（成肌诱导剂）。形态学检测肌管形成情况；MTT法检测成肌细胞增殖；流式细胞仪检测成肌细胞凋亡；Western Blot检测肌调节标志物Pax7、MyoD、Myf5、Myf6和Myogenin蛋白；免疫荧光检测Desmin、Pax7、MyoD的表达。 结果：①结果显示成肌细胞的活力对冲击波刺激呈现剂量依赖性，能流密度高于0.16 mJ/mm2时细胞的存活率降低，凋亡率高于空白组（P<0.05），能流密度为0.08 mJ/mm2时，冲击次数为500时细胞最高活力。②免疫荧光结果显示冲击波可促进MyoD蛋白表达升高，降低Pax7的表达。③Western Blot结果显示冲击波可促进MyoD、Myf5、Myogenin、Myf6蛋白表达水平显著增高（P<0.05），Pax7蛋白表达随着时间的延长呈递减趋势（P<0.05）。MyoD蛋白在实验组和对照组随着时间延长蛋白表达水平都逐渐升高，且实验组的表达水平均高于对照组（P<0.05）。④细胞形态学观察肌管融合结果显示，成肌细胞在冲击波刺激下呈现多核，实验组、对照组与空白组相比差异具有显著性意义（P<0.05）。 结论：本实验证实了体外冲击波对成肌细胞的生物学功能有影响，可提高成肌细胞的活力，促进其向肌分化。

Abstract:

      Abstract Objective：To investigate the effect of extracorporeal shock wave on the biological function of myoblasts，to lay the foundation for further research on the role of shock wave in skeletal muscle injury in vivo，and to provide theoretical basis for clinical application of shock wave therapy. Method：We isolated the primary cultured myoblasts and stimulated them with shock waves. The myoblasts were divided into blank group，experimental group (shock wave stimulation) and positive control group (myoblast inducer). The changes of myotubes formation were detected by morphological method； MTT method was used to detect the proliferation of myoblasts； Flow cytometry was used to detect the apoptosis of myoblasts；Western Blot was used to detect the expression of MyoD， Myf5， Myf6 and Myogenin proteins；Immunofluorescence was used to detect the expression of Desmin， Pax7 and MyoD. Result：①The viability of myoblasts exhibited a dose-dependent relationship with shock wave stimulation. The cell survival rate decreased when the energy flow density was above 0.16 mJ/mm2，with the higher apoptotic rate than the blank group(P<0.05). The highest cell viability was appeared when 500 of the number impacts and 0.08 mJ/mm2 of the energy flow density. ②Immunofluorescence showed that the shock wave could promote the increase of MyoD protein expression and decrease the expression of Pax7. ③Western Blot results showed that the shock waves could significantly increase the expression levels of MyoD，Myf5， Myogenin， and Myf6 (P<0.05). Pax7 protein expression decreased with time prolonging， and the differences were most obvious between the 1d and 3d groups(P<0.05). The expression level of MyoD protein in the two groups gradually increased with time prolonging，and the expression level of the shock wave group was higher than that of the blank control group，and there were differences between the two time periods(P<0.05).④The morphological observation of myotube fusion showed a multiple nuclei myoblast under shock wave stimulation，with obvious myotube formation. After shock wave，the experimental group and the positive control group showed statistically different myotube fusion indexes when compared with the control group (P<0.05). Conclusion：This experiment confirms that the extracorporeal shock wave has an effect on the biological function of myoblasts by increasing the viability of myoblasts and promoting their differentiation into muscles.