摘要
背景:多裂肌的损伤、萎缩是临床各种腰背痛及功能障碍的主要来源之一,肌卫星细胞是肌肉干细胞,对肌肉的损伤修复起至关重要的作用。但目前尚缺少有关多裂肌卫星细胞分离培养方面的研究。目的:采用单根肌纤维法分离大鼠多裂肌卫星细胞并进行鉴定。方法:在体分离大鼠多裂肌,采用单根肌纤维法获得多裂肌卫星细胞,经反复差速贴壁法纯化,观察其形态变化并绘制细胞增殖曲线;以分化培养基进行诱导,观察其成肌分化情况;并采用免疫荧光法鉴定多裂肌卫星细胞标志蛋白Pax7、Myod、Desmin的表达。结果与结论:(1)单根多裂肌纤维经培养72 h后,可见肌纤维周围有大量长梭形或纺锤形细胞贴壁;(2)采用差速贴壁法纯化后,可见原代细胞呈圆形,高折光性,重新接种后24-48 h可贴壁,其生长呈"S"型;(3)以分化培养基诱导可出现较大肌管;(4)免疫荧光鉴定可见Pax7、Myod在肌卫星细胞的细胞核中为阳性反应,Desmin在细胞质中为阳性反应;(5)结果表明,单根肌纤维法可成功分离大鼠多裂肌卫星细胞,可用于未来多裂肌再生修复方面的研究。
BACKGROUND:Atrophy and damage of multifidus muscles mainly result in various low back pains and dysfunctions.Muscle satellite cells,which belong to muscle stem cells,play an important role for muscle regeneration after injury.However,little is reported on the isolation and culture of multifidus muscle up to now.OBJECTIVE:To isolate and identify multifidus satellite cells of rats using single muscle fiber method.METHODS:Multifidus muscles were isolated in vivo and multifidus satellite cells were obtained by single muscle fiber method.Then the cells were purified by repeated differential attachment.Morphological changes of the cells were observed and the proliferation curve was made thereafter.Moreover,myogenic differentiation of the isolated cells cultured in differentiation medium was analyzed and marker proteins of multifidus satellite cells(Pax7,Myod,Desmin)were characterized by immunofluorescence.RESULTS AND CONCLUSION:A large number of fusiform or spindle cells were seen around the muscle fibers after 72 hours of culture.Round cells with high refraction were visible after being purified by repeated differential attachment,and the cells adhered after 24-48 hours of re-inoculation exhibited an“S”shaped growth curve.Large myotubes formed in the differentiation medium.Immunofluorescence findings showed that Pax7 and Myod were positively expressed in muscle satellite cell nuclei,while Desmin was positively expressed in the cytoplasm.These findings reveal that multifidus satellite cells can be successfully separated by single muscle fiber method,which can be used for regeneration of multifidus muscles in the future.
作者
刘通
于佳妮
陈玉佩
陈欢
邹德辉
晏珺
卢宗孝
刘悦
张莉
霍则军
Liu Tong;Yu Jia-ni;Chen Yu-pei;Chen Huan;Zou De-hui;Yan Jun;Lu Zong-xiao;Liu Yue;Zhang Li;Huo Ze-jun(Department of Acupuncture and Rehabilitation,Guangdong Second Hospital of Traditional Chinese Medicine,Guangzhou 510095,Guangdong Province,China;Department of Rehabilitation,Guangdong Hospital of Traditional Chinese Medicine,Guangzhou 510120,Guangdong Province,China;School of Acu-moxibustion and Tuina,Beijing University of Traditional Chinese Medicine,Beijing 100029,China;Department of Acupuncture and Moxibustion,Jiangsu Province Hospital,Nanjing 210000,Jiangsu Province,China;Department of Chinese Medicine,Peking University 3rd Hospital,Beijing 100191,China)
出处
《中国组织工程研究》
CAS
北大核心
2018年第25期4065-4070,共6页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金青年基金(81704179
81503641)
国家自然科学基金面上项目(81574052)~~
