盆底肌肉复合体重建及修复对改善先天性肛门直肠畸形术后排便功能的作用的研究

The maldevelpopment of the pelvic floor muscle and aberrant innervation in anorectal malformations plays an important role in the postoperative bowel dysfunction. Researches have confirmed that the satellite cells plays an important role in the process of normal pelvic floor muscle. The aim of the project is to study the embryonic development of satellite cells in pelvic floor muscle and their microenvironment in normal and anorectal malformations fetal rats , and to explore the perhaps mechanisms of the pelvic floor muscle abnormalities in anorectal malformations. At the same time, we utilize the character of the muscle satellite cells can be cultured in vitro and can survive, migrate and differentiate. We plant the satellite cells as seed cells in the absorbable stents of tissue engineering. After successful culture in vitro, we engraftment the stents to the pelvic floor muscles, to further explore the tissue engineering approach to the repair of pelvic floor muscle in anorectal malformatios. We want to look for some new ideas and methods to the clinical treatment of postoperative bowel dysfunction of anorectal malformations.

盆底肌发育及神经支配异常是先天性肛门直肠畸形术后排便功能障碍的重要原因之一，研究证实卫星细胞在正常盆底肌的胚胎发育过程中起着重要作用。本课题旨在研究正常及肛门直肠畸形胎鼠盆底肌卫星细胞及其微环境的胚胎发育过程，探索肛门直肠畸形盆底肌异常发育的机制。另外我们根据肌卫星细胞可以在体外培养环境中存活、迁移和分化的特性，利用组织工程的三维可吸收支架，以卫星细胞为种子细胞，体外培养成功后移植入体内，观察移植后效果，进一步探讨组织工程方法修复先天性肛门畸形发育不良盆底肌的方法，为将来进一步临床治疗提供新思路和新方法。

本研究应用乙烯硫脲（ethylenethiourea, ETU）致畸建立的Wistar大鼠肛门直肠畸形（anorectal malformations, ARMs）动物模型，验证了Wnt5a, Wnt3a, BMP4,BMP7及Notch1基因在正常胎鼠及肛门直肠畸形胎鼠盆底横纹肌复合体（striated muscle complex, SMC）发育过程中的表达情况，并同时验证了GHRL及GHSR在正常胎鼠及肛门直肠畸形胎鼠肛门直肠区的表达情况。结果提示Wnt5a, Wnt3a, BMP4及BMP7基因均在正常胎鼠及肛门直肠畸形胎鼠肛门直肠区SMC发育过程中表达存在差异；Notch1基因在正常胎鼠及肛门直肠畸形胎鼠肛门直肠区SMC发育过程中表达无明显差异。在E15d和E17d，正常胎鼠和肛门直肠畸形胎鼠在肛门直肠上皮处的GHRL和GHSR表达水平有显著差异，E17d和E19d则无明显差异。另一部分细胞实验，提取骨骼肌卫星细胞，经过体外培养增殖，绿色荧光蛋白标记后种植入组织工程可吸收支架内，结果提示，用GFP-腺病毒转染肌卫星细胞，当转染48h时荧光强度最高。利用组织工程支架将卫星细胞移植于ARMs胎鼠盆底肌区域，观察卫星细胞的体内分化方向及能力，结果发现移植的肌卫星细胞主要位于发育异常的SMC附近，而且表现为不同的细胞形状，如圆形、细长形和纺锤形等。GFP及Desmin双重表达阳性，表明eGFP标记的肌卫星细胞分化为成肌细胞；而且，GFP及Myosin 双阳性，提示eGFP标记的肌卫星细胞分化为成熟骨骼肌细胞。