大鼠骨骼肌去细胞外基质水凝胶联合骨骼肌干细胞对骨骼肌缺损的修复作用  

Repair of skeletal muscle defects by skeletal muscle acellular matrix hydrogel combined with skeletal muscle stem cells in rats

作　　者：祝孟海 秦本刚[1] 杨羿[1] 杨建涛[1] 顾立强[1] ZHU Menghai;QIN Bengang;YANG Yi;YANG Jiantao;GU Liqiang(Department of Hand,Trauma and Microsurgery,The First Affiliated Hospital of Sun Yat-sen University,Guangzhou 510080,China)

机构地区：[1]中山大学附属第一医院显微创伤手外科,广州510080

出　　处：《中华显微外科杂志》2020年第5期473-480,共8页Chinese Journal of Microsurgery

基　　金：国家自然科学基金(81871787,81601057);广东省自然科学基金(2018A030310254,2015A030310350,2019A1515012057);广东省医学科技研究基金(A2017190)。

摘　　要：目的在大鼠骨骼肌缺损模型上,探讨骨骼肌去细胞外基质(ECM)水凝胶联合骨骼肌干细胞对骨骼肌缺损的修复作用。方法2018年5月至2020年1月,采用5种方法将大鼠骨骼肌脱细胞,使用苏木精-伊红(HE)染色、DAPI染色评估骨骼肌脱细胞情况,选出最优策略制成水凝胶。采用HE染色、Masson染色、扫描电镜(SEM)等对水凝胶的结构、成分进行分析。将骨骼肌干细胞植入包被有水凝胶的培养皿,MTT法检测细胞生长情况;构建大鼠骨骼肌缺损动物模型,用HE染色检测骨骼肌生长,免疫荧光检测Pax7和MyoD的表达情况,电刺激对各组大鼠骨骼肌组织修复后功能进行评估;使用t检验和单因素方差分析进行数据分析。结果本研究发现了一种对骨骼肌结构损伤小并且彻底的脱细胞的方法,然后将其制备成水凝胶;Masson染色、SEM表明水凝胶保留了骨骼肌的基本结构和部分生物因子;MTT检测表明该水凝胶能够促进骨骼肌干细胞的增殖。大鼠骨骼肌缺损实验表明大鼠骨骼肌干细胞联合水凝胶治疗组比力为(9.00±2.10)N/cm^2,分别与未修复组和水凝胶组[分别为(4.06±1.12)N/cm^2和(5.00±1.60)N/cm^2]比较,差异有统计学意义(P<0.05),联合组有更好的效果。结论骨骼肌ECM水凝胶有较好的生物学性能,该水凝胶联合干细胞有望能成为一种修复骨骼肌缺损的有效方法。Objective To explore the therapeutic outcome of rat skeletal muscle extracellular matrix(ECM)hydrogels blended with skeletal muscle stem cells for skeletal muscle defects in a rat model.Methods From May,2018 to January,2020,5 methods were employed to obtain ECM from rat skeletal muscle.The resulting ECM was evaluated using haematoxylin and eosin(HE)staning and 4-6-diamidino-2-phenylindole(DAPI)staining.The components and structure of hydrogel were assessed by Masson staining and scanning electron microscopy(SEM).Skeletal muscle stem cells were then blended in the hydrogel and MTT was used to observe the growth of cells.Rat skeletal muscle defect models were established and HE staining was used to examine the growth of injured skeletal muscle in each group.Expression of Pax7 and MyoD were detected by immunofluorescence in each group.The function of skeletomuscular tissue was evaluated by electrical stimulation in each group.T test and ONE-WAY analysis of variance were used for data analysis.Results In this study,a method that caused little damage to skeletal muscle structure and completely removed the cell components was found.This method was employed in the preparation of hydrogel.Masson staining and SEM showed that hydrogel retained the basic structure and some biological factors of skeletal muscle.MTT test showed that hydrogel promoted the proliferation of skeletal muscle stem cells.The experiments in rat skeletal muscle defect models showed that the group treated with stem cells[Specific force was(9.00±2.10)N/cm^2 achieved better outcomes than the other groups(4.06±1.12)N/cm^2 and(5.00±1.60)N/cm^2 in unrepaired group and hyclrogel group,respectively](P<0.05).Conclusion The results indicated that ECM hydrogel has a good biocompatibility.Furthermore,it was observed that rat skeletal muscle ECM hydrogels blended with skeletal muscle stem cells promoted the repair of muscle defect in a rat model,which would offer a promising strategy for functional muscle repair.

关 键 词：骨骼肌干细胞 去细胞外基质 骨骼肌缺损 大鼠 

分 类 号：R318.08[医药卫生—生物医学工程]