【作者】 乔东访；

【导师】 朴英杰；

【作者基本信息】 中国人民解放军第一军医大学 ， 人体解剖与组织胚胎学， 2003， 硕士

【摘要】 骨骼肌损伤或缺损在临床的创伤治疗中较为常见，为了更好地探讨骨骼肌缺损的修复途径，本实验将自体的骨髓MSCs进行成肌诱导分化后复合HHK材料，对较大面积的骨骼肌缺损修复进行了研究。 在骨髓MSCs分离和体外扩增实验中，通过对细胞的形态学观察、生长曲线测定和细胞周期分析等，结果表明，在采用较低浓度血清的培养条件下，骨髓MSCs具有较好的生长和增殖特性，可以为后续体外诱导实验提供状态良好且足够数量的细胞来源。 在将体外扩增后的骨髓MSCs进行成肌诱导分化的实验中，经RT-PCR检测成肌基因的激活、流式细胞仪和免疫组化检测成肌细胞特异蛋白(结蛋白)的表达，结果表明浓度为10μmol／L的5-Aza可以使上述细胞发生成肌分化，诱导后可获得含较高比例成肌细胞的细胞群体，能够满足用于植入体内对缺损骨骼肌进行修复的需要。 在成肌诱导后的自体骨髓MSCs与HHK材料复合植入骨骼肌缺损动物模型实验中，首先对HHK材料在骨骼肌组织内的降解过程进行了研究，经光、电镜和Ub免疫组化观察，结果表明，植入后第3～6w为降解高峰期，到第9w材料的降解基本完成；在整个降解的过程中首先是通过体内的Ub系统将HHK材料降解成微小的碎片，再由巨噬细胞等通过吞噬完成清除。HHK材料作为植入的空间支架，因其具有一定的方向性，能够满足使植入的细胞按骨骼肌纤维走向进行生长的要求，实验研究发现其对植入细胞的生长方向具有较好的诱导作用。 骨骼肌缺损修复实验中分三个实验组进行比较，单纯植入HHK材料组、未诱导自体骨髓MSCs与HHK材料复合植入组和成肌诱导分化后自体骨髓MSCs复合HHK材料植入组。通过光、电镜形态学和骨骼肌肌球蛋白免疫组化观察，结合细胞示踪，结果表明，从植入部位新生骨骼肌纤维出现的时间以及密度分析，成肌诱导后自体骨髓MSCs复合HHK材料植入组获得的修复效果均优于前两组。更多还原

【Abstract】 The damage and defect of skeletal muscle is common surgery. In order to investigate the repair of defect skeletal muscle, the autogenous bone marrow derived mesenchymal stem cells （MSCs） were induced to myoblasts at first. Then the compound of the myoblasts and human hair keratin （HHK） was used for the repair of the defect skeletal muscle.The bone marrow derived MSCs possessed of the ability of proliferation in high rate and could grow in stable, in the lower concentration of serum, through the observation of morphology and the assay of proliferation capability and cell cycle. In this condition, we could obtain sufficient cells for the next inducing experiments.In the process of inducing the bone marrow derived MSCs to myoblasts in vitro, l0μmol/L 5-azacytidin was effective, through the detection of the activation of myoblast determination genes with RT-PCR, and the expression of skeletal muscle desmin with flow cytometry and immiinohistochemistry. The cell population containing higher proportion of myoblasts, therefore, it could be used for the next implantation.The degradation progress of HHK scaffold material was observed through the routine morphological and ubiquitin immunohistochemical study, after the implantation of the compound of the induced myoblasts and HHK scaffold material. The results made clear that the degradation was in the peak after implantation 3-6^th week, and accomplished in about 9th week. The HHK scaffold material was degraded into small particles by ubiquitin system at first, and then those particles phagocytosed by macrophages. At the same time, because of directivity of HHK scaffold material, from our works, it could lead the cells to grow in parallel with the muscle fiber.There were three groups for the comparison of the repair results: HHKscaffold material implantation group （1st group）, the compound of the autogenous bone marrow derived MSCs and HHK scaffold material implantation group （2nd group）, and the compound of the autogenous induced myoblasts and HHK scaffold material implantation group （3rd group）. Among the three groups, the repair effectiveness of the 3rd group was the best than the others, on the basis of the time of regeneration myofibers formation and it density, through the morphological and myosin immunohistochemical study更多还原