VEGF基因修饰脂肪干细胞成肌分化膜片碎片化注射治疗压力性尿失禁的实验研究

Stress urinary incontinence (SUI) is a common urological disease in female patients. Currently, the surgical treatment may cause several complications, such as organ/vascular injury and infection. And more importantly, the mechanism of urethral sphincter deficiency cannot be essentially improved. Recently, the tissue engineering technology provide an new therapeutic strategy for SUI. However, how to ensure the cell planting after transplantation, maintain the survival rate after cell transplantation and fundamentally improve the function and structural defects of the urethral sphincter has become the key point for the long-term efficacy of SUI. In this study, rat autogenous adipose derived stem cells (ADSCs) are used as seed cells. Then, we promote vascularization by lentivirus transfection of vascular endothelial growth factor (VEGF) gene, stimulate extracellular matrix secretion by vitamin C, induce the ADSCs into myoblasts differentiation by 5-azacytidine and horse serum, establish VEGF gene modified myoblasts differentiated cell sheet, then the myoblasts differentiated cell sheet was cut into pieces and directly injected into the urethral sphincter to treat SUI. The cell sheet pieces can support urethra by extracellular matrix (ECM), promote the cell planting with the extracellular matrix adhesion in the short term, and in the long term, urethral sphincter can be enhance through the vascular muscle cells regeneration. The urodynamic and urethral sphincter histological examination will be used for evaluating the outcome of this therapy. In conclusion, we aim to investigate the value of VEGF gene modified myoblasts differentiated cell sheet pieces injection in the SUI treatment.

压力性尿失禁（SUI）是女性泌尿系统常见疾病，目前临床手术治疗容易引起盆腔脏器损伤和感染等并发症，且不能改变尿道括约肌功能减退的本质，而组织工程技术提供新的治疗途径，但如何促进移植后细胞定植，维持细胞移植后存活率且从根本上纠正尿道括约肌功能和结构缺陷，已成为影响SUI长期疗效关键。本研究以大鼠自体脂肪干细胞（ADSCs）作为种子细胞，慢病毒转染血管内皮生长因子（VEGF）基因使其过表达促血管化，采用维生素C刺激细胞外基质（ECM）分泌，马血清和5-氮杂胞苷诱导成肌分化，构建VEGF基因修饰成肌分化细胞膜片，并将其碎片化注射至膀胱颈尿道周围，短期内通过ECM支撑稳定尿道，同时ECM黏附性强可促进细胞定植，远期内通过血管化肌细胞扩增，增强肌力，从而治疗SUI；通过尿道括约肌局部组织学和尿流动力学检测，分析治疗效果，探索VEGF基因修饰ADSCs成肌分化细胞膜片碎片化注射治疗SUI的应用价值。

压力性尿失禁（SUI）是泌尿外科常见疾病，表现为运动和咳嗽等“压力”条件下尿液非自愿泄漏，女性SUI发生率约为22%。尿道填充剂注射是一种治疗轻中度SUI的微创方式，具有安全、低风险和适用人群广泛的优势。然而，传统填充剂多为非天然来源，免疫原性高。更为重要的是，传统填充剂不能促进肌组织再生，从而导致远期效果欠佳。在本研究中，我们开发一种新型注射填充剂，即脂肪干细胞膜片脱基质（ADSC-ECM）片段，并探索其SUI治疗有效性。首先制备ADSC细胞膜片，利用Triton X-100法脱细胞处理，制备ADSC-ECM，检测其中蛋白胶原纤维未挛缩和破坏，ADSC膜片和ADSC-ECM中VEGF、bFGF或PDGF水平之间没有显著差异，P>0.05，丰富的生长因子含量有助于细胞粘附、迁移、增殖和分化。将ADSC-ECM灭菌并切成片段（长度=416±49μm）便于注射器注射。为了定位移植的ADSC-ECM片段，用超微超顺磁纳米氧化铁颗粒（USPIO）标记ADSC-ECM，设置正常组、ADSC-ECM组和生理盐水组，注射至SUI大鼠模型后尿道及膀胱颈周围，分别予以1周和4周体内监测尿动力学变化，注射后1周，ADSC-ECM组的LPP（37.8±2.9 cmH2O）恢复到接近正常水平（40.8±2.0 cmH2O），高于生理盐水组（22.0±4.2 cmH2O）（P <0.05）。注射后4周，生理盐水组（23.8±3.8 cmH2O）的LPP显着低于正常组（42.7±2.4 cmH2O）（P<0.05）。ADSC-ECM组的LPP保持较高水平（35.5±3.4 cmH2O），与正常组相比无显着差异，P>0.05。分别于体内注射1周和4周后，处死实验动物，组织学染色表明ADSC-ECM片段注射尿道后1周即可与周围组织融合。移植4周后，宿主细胞渗透至ADSC-ECM片段注射区域内。此外，肌球蛋白免疫荧光染色证实移植ADSC-ECM片段区域形成新的平滑肌组织。同时，注射型ADSC-ECM具有高度生物安全性，不会在体内主要器官蓄积，不影响大鼠肝肾功能。上述结果表明，注射ADSC-ECM片段治疗SUI比传统填充剂更具有优势，具有较大临床应用潜力。本研究完成ADSC-ECM片段注射在SUI治疗中的应用价值和可能作用机制的相关探索，为该项技术运用于临床治疗打下坚实基础。