载anti-Sca-1与bFGF的功能化UBM支架捕获自体干细胞原位修复盆底支持结构的研究

Pelvic support structural damage is the leading cause of pelvic organ prolapse (POP). At present, polypropylene -based filler material is a common means of treating POP , but it can not rebuild the pelvic support structures from the function . Early discovery : bladder acellular matrix (UBM) is the most numerous biological scaffold material suitable for pelvic floor repair . Recent studies have found that , UBM can induce tissue regeneration , and the mechanism that homing of stem cells to promote tissue repair UBM area , but there is still a local POP UBM treatment cell colonization fewer long-term efficacy is poor. Therefore , this study in the early about UBM, anti-Sca-1 bFGF research base and on the use of Sulfo-SMCC chemical crosslinking method to build anchors anti-Sca-1 and bFGF function of UBM stand by in vivo experiments in vitro its capture ability of autologous pluripotent stem cells were observed before and after the mechanical characteristics of the pelvic support structures repaired. Expectations: both capture a large number of autologous stem cell proliferation in the spatial structure of the stent , but also on the basis of its structure to further promote mature , replacement therapy is to develop a new strategy pelvic organ prolapse tissue engineering materials.

盆底支持结构损伤是导致盆腔器官脱垂（POP）的主要原因。目前，以聚丙烯材料为主的填充材料是治疗POP的常用手段，但其无法从功能上重建盆底支持结构。前期发现：脱细胞膀胱基质（UBM）是众多生物支架材料中最适合盆底修复的材料。新近研究发现，UBM在机械填充的同时，能诱导组织再生，其机制在于干细胞归巢到UBM区域促进组织修复，但UBM治疗POP仍存在局部定植细胞较少,远期疗效差的问题。因此，本研究拟在前期有关UBM、anti-Sca-1与bFGF研究基础上,采用Sulfo-SMCC化学交联方法构建锚定anti-Sca-1与bFGF的功能化UBM支架，通过体内体外实验验证其捕获自体多能干细胞能力，观察盆底支持结构修复前后力学特征。期望：既能捕获自体干细胞在支架空间结构内大量增殖，又能在此基础上进一步促进其结构成熟，为盆腔器官脱垂组织工程材料替代治疗开拓新的策略。

盆腔器官脱垂(pelvic organ prolapse,POP)是一种常见妇科疾病, 其发病率随我国人口老龄化而逐年增加，严重影响患者健康和生活质量。目前使用补片临床常用补片进行治疗，但远期效果不好。本课题将负载抗sca-1抗体（anti-sca-1）和碱性成纤维细胞生长因子（basic fibroblast growth factor, bFGF）结合到脱细胞膀胱基质（urinary bladder matrix, UBM）生物支架上，探讨其作为新型盆底修复材料的可能性。方法 通过双特异性化学交联试剂4-(N-马来酰亚胺甲基)环己烷-1-羧酸磺酸基琥珀酰亚胺酯钠盐（sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate, sulfo-smcc）将anti-sca-1和 bFGF特异性结合到UBM上，得到anti-sca-1/bFGF-UBM生物支架。通过扫描电子显微镜观察生物支架表面结构，CCK-8（cell counting kit-8）间接检测体外富集sca-1阳性细胞情况，免疫荧光观察支架诱导小鼠骨髓间充质干细胞（mouse mesenchymal stem cells， mMSCs）分化成熟情况。结果 扫描电子显微镜显示anti-sca-1和bFGF能特异性的结合到UBM上。荧光强度测定anti-sca-1和bFGF明显高于对照组（分别为7.304±0.103 vs 1.482±0.136,12387.033±509.432 vs 6855.506±114.702， P＜0.05）。anti-sca-1/bFGF-UBM生物支架富集的sca-1阳性细胞明显较对照组多（1.087±0.049 vs 0.348±0.004，P＜0.05）。免疫荧光提示anti-sca-1/bFGF-UBM生物支架可以促进mMSCs向平滑肌细胞分化。结论 anti-sca-1/bFGF-UBM生物支架显示出了良好的生物性能，既能富集sca-1阳性细胞，又能进一步促进细胞分化成熟，有望成为盆底支持结构修复的候选材料。