肌球蛋白轻链在肌肉再生中作用的体外实验研究  

AN EXPERIMENTAL STUDY OF THE ROLE OF MYOSIN LIGHT CHAIN IN MYOGENESIS IN VITRO

作　　者：张素珍[1] 解慧琪[2] 徐永[3] 李秀群[2] 邓力[2] 陈晓禾[2] 邱琳[2] 杨志明[2] 

机构地区：[1]四川大学生命科学学院,成都610041 [2]四川大学华西医院生物治疗国家重点实验室.干细胞与组织工程研究室 [3]四川大学华西医院肿瘤生物治疗研究室

出　　处：《中国修复重建外科杂志》2008年第6期753-758,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(30670734);四川省科技厅青年基金资助项目(042Q026-038)~~

摘　　要：目的应用不同来源的成肌细胞(myoblast,Mb)研究肌球蛋白轻链(myosin light chain,Myl)在肌肉再生中的作用,为进一步了解Myl的生理功能提供依据。方法3周龄健康雄性C57BL/6小鼠12只。采用酶消化法和Preplate技术分离纯化小鼠眼外肌、膈肌和腓肠肌Mb(eMb、dMb和gMb),进行传代培养。采用RT-PCR和Western blot法检测第1代细胞Myl的表达,亚甲基蓝法检测细胞增殖能力,倒置相差显微镜观察肌管形成情况。采用浓度为1、2、4、8、16 ng/mL Myl单克隆抗体分别处理3种细胞(实验组),与未处理的细胞比较(对照组),观察细胞增殖能力及肌管形成的变化。结果培养24 h的eMb、dMb和gMb均检测到Myl1、Myl4 mRNA和Myl蛋白表达,且eMb的表达水平显著低于dMb和gMb(P<0.01),dMb和gMb间差异无统计学意义(P>0.05);3种细胞均未检测到Myl2和Myl3 mRNA表达。eMb的增殖速度高于dMb和gMb(P<0.01);eMb和dMb、gMb分别在接种后40 h和16 h出现肌管;第6天eMb肌管数为(137.2±24.5)/视野,显著高于dMb的(47.6±15.5)/视野和gMb的(39.8±5.1)/视野(P<0.01),后两者差异无统计学意义(P>0.05)。Myl抗体作用后,3种细胞实验组各浓度吸光度值均显著高于对照组(P<0.05),表现浓度依赖性;dMb实验组和对照组16 h肌管数分别为(48.2±7.1)/孔和(23.4±4.9)/孔,6 d肌管数分别为(40.6±10.2)/视野和(63.1±6.1)/视野,两组间差异均有统计学意义(P<0.01)。结论Myl可能通过促使Mb终末分化,负性影响Mb增殖而在肌肉再生中发挥一定作用。Objective To investigate the role of myosin light chain （Myl） in myogenesis in vitro. Methods The extraocular muscle, diaphragm and gastrocnemius muscle myoblasts （eMb, dMb and gMb） were isolated and purified from 12 3-week-old C57BL/6 mice by using the enzyme digestion and Preplate technique, and then were subcultivated. The Myl expression in Mb was detected by RT-PCR and Western blot analysis; the Mb proliferation activity was tested by methylene blue assay, and the myotube formation was observed. After anti-Myl antibody （1, 2, 3, 8, 16 ng/mL） was induced in the Mb culture （experimental group）, the ability of proliferation of myoblasts and the myotube formation were identified. Meanwhile, the Mb which was cultured without anti-Myl antibody was indentified as the control group. Results The results of RT-PCR and Western blot analysis showed that Myll and Myl4 mRNA and Myl protein were expressed in eMb, dMb and gMb at 24 hours after seeding, and their expression level were lower in eMb than in dMb and gMb （P 〈 0.01）, and the latter two did not show any significant difference （P 〉 0.05）. Myl2 and Myl3 mRNA was not detected in these three myoblasts. The proliferation assay showed that the eMb proliferated faster as compared with dMb and gMb （P 〈 0.01）. eMb began to yield myotubes at 40 hours after seeding and dMb and gMb at 16 hours after seeding. At 6 days, the number of myotubes derived from eMb was （137.2 ± 24.5）/field, which was significantly larger than that of myotubes from dMb [（47.6 ± 15.5） / field ] and gMb [（39.8 ± 5.1）/ field ] （P 〈 0.01）. There was not statistically significant difference between the latter two groups （P 〉 0.05）. After the antibody treatment, the absorbency values of the eMb, dMb and gMb in the experimental groups at each antibody concentration point were significantly higher than those in the corresponding control groups （P 〈 0.05）, and the dose-dependent way was performed. The numbers of myotubes from dMb at 16 hours we

关 键 词：肌球蛋白轻链 成肌细胞 增殖 肌肉再生 体外研究 小鼠 

分 类 号：Q51[生物学—生物化学]