摘要目的 研究成肌细胞移植重建盆底横纹肌复合体的可行性,为肛门直肠畸形盆底肌发育不良探索新的治疗方法.方法 提取新生大鼠骨骼肌卫星细胞进行体外培养,分化形成成肌细胞,转染携带绿色荧光蛋白的腺病毒后种植到建立的横纹肌复合体去细胞空支架中,体外培养2d后支架移植回盆底在体内生长4周,应用电镜及免疫荧光化学染色检测成肌细胞在体内分化融合形成骨骼肌纤维的情况.结果 卫星细胞分离后,贴壁较早的成肌细胞增殖较快,很快分化融合成多核肌管,传代次数有限;贴壁较晚的细胞中有较多呈球形细胞,表达卫星细胞特异性标记Pax7,其增殖较慢,传代次数较多.成肌细胞Desmin表达阳性,分化成肌管后表达Myosin.腺病毒转染后转染率达98.6%.建立的空支架Laminin表达阳性,由以胶原纤维成分为主的基底膜管组成,成肌细胞种植后细胞附着于支架,并见细胞突起,支架体外培养1周后细胞仍存活.移植后4周成肌细胞分化形成骨骼肌,在球海绵体肌后方形成肌束,但未形成弹弓样结构的横纹肌复合体,GFP及Myosin表达阳性,肌纤维周围可见微血管形成,并且检测到Pax7表达阳性的卫星细胞.结论 卫星细胞经体外分离培养后移植至盆底能很好地分化形成肌纤维,可以作为修复发育不良的盆底肌的细胞来源.

abstractsObjective To investigate the feasibility of myoblast transplantation in the regeneration of striated muscle complex, explore a potential treatment strategy to improve the pelvic floor muscle in anorectal malformations. Methods Skeletal muscle satellite cells obtained from the hindlimbs of neonatal Wistar rats were purified and cultured. eGFP labeled myoblasts were seeded into the acellular matrices, obtained by hypotonic-detergent treatment of striated muscle complex. After 2 d culture in vitro, myoblast-seeded matrices were implanted in situ into the pelvic floor. 4w after surgery, the matrices were harvested and observed with microscopy and immunofluorescence. Results Cultured cells were characterized as myoblasts by the presence of specific antigens Desmin and Myosin with immunocytochemical staining. Two subpopulations were identified in myoblasts, the earlier preplated cells proliferated and differentiated quickly, terminal differentiated into myotube rapidly, with limited passage ability; while the later preplated subclone still remain stem cell-like properties, positively expressed Pax7, a satellite cell marker, and divided slowly. 98. 6% cells were viral integrated 24h after transfeetion of eGFP-adenovirus as detected by flow cytometry. The acellular matrices mainly composed of basal membrane tube which were Laminin positive; at 48h from seeding, matrices showed many round myoblasts attached; the cultures remained viable until the 7th day, and fused myoblasts were seen. 4w after transplantation, muscle bundles can be seen behind M. bulbocarvernosus, but failed to form a sling-like structure of SMC. The myoblasts differentiated into myofibers, which were GFP and Myosin double positive staining, neovascularization were evidenced as vWF positive expressing vascular endothelial cells were observed, and Pax7 positive satellite cells were founded outside the sarcolemma of muscle fibers. Conclusions The satellite cells, after being implanted into the pelvic floor, showed strong proliferation and differentiation ability, they arefit to construct the cell bank of tissue engineering in rernolding the maldeveloped pelvic floor muscle. Further researches are needed to identify strategies to improve and maintain the structural and functional integrity of implants.