空间梯度分布的RGD多肽-丝素蛋白支架在尿道重建中的实验研究

Urethral stricture is a common ailment presenting to urologists, with high recurrence rate. Urethroplasty with autologous tissue confronts two challenges: limited sources and many complications. Therefore, tissue engineering techniques are promising in urethral reconstructions. Silk fibroin (SF) shows distinct advantages in tissue regeneration, biocompatibility and so on. However, SF scaffold currently used shows two weak points which could lead to poor functional organ performance. First, it’s not able to mimic the gradient spatial micro-structure of natural urethral extracellular matrix (ECM). Second, it’s unfavorable for cell adhesion, due to lack of cell adhesion molecules. To overcome these two shortcomings, we will optimize SF scaffold with gradient spatial micro-structure and then combine it with RGD polypeptide. Cell adhesion, biocompatibility and biodegradability rate will be detected in vitro and vivo test with mouse model. Then we will develop tissue-engineered mucosa membrane with optimized SF. Urethral reconstruction with tissue-engineered RGD peptides-SF scaffold will be investigated in dog urethral stricture models. The repair and function of urethra will be evaluated by retrograde urethrography, histologic and immunohistochemical analysis. Our study is the first to propose that the SF scaffold with the gradient spatial micro-structure like the natural urethral extracellular matrix) and improve it by combining RGD-peptide. We hypothesized that this research would create a better plan in urethral reconstruction and provide new ideas for the repair of other hollow organs.

尿道狭窄是泌尿外科常见且易复发的疾病。目前所用的自体组织替代尿道成形术面临组织来源有限和并发症较多的问题，因此组织工程技术在尿道重建上富有前景，而理想的支架材料是其重要基础。丝素蛋白（SF）在组织再生、生物相容性等方面有明显优势，但目前使用的SF支架不能模拟梯度分布的天然尿道细胞外基质，同时缺乏生物识别位点不利于细胞黏附，导致重建的尿道结构不稳定。为解决以上问题，本研究构建空间微细结构梯度分布SF支架并与生物黏附多肽RGD结合，通过体外和小鼠体内实验观察细胞黏附生长情况、生物相容性和材料降解速率；利用优化的RGD多肽-SF支架体外构建组织工程粘膜，并修复重建犬尿道狭窄模型，术后全面评价尿道功能及支架降解吸收情况。我们首次在尿道重建中提出梯度分布概念（模拟天然尿道细胞外基质），并结合RGD多肽修饰SF支架材料，探索更合理的组织工程尿道修复方案，也为其他中空器官的修复提供新思路。

先天性畸形、外伤、肿瘤等导致的尿道狭窄是泌尿外科常见的疾病，以往多采用临近皮瓣（阴茎、阴囊皮肤）、游离粘膜移植物（舌粘膜、颊粘膜、膀胱粘膜等）进行手术修复，自体材料有其局限性，如术后并发症、材料有限、供体部位的相关并发症等等。因此，我们寻找一种新的材料来修复尿道狭窄。本研究中我们采用模拟天然尿道细胞外基质的双层结构，内层由PLLA/poly(L-lactide-co-e-caprolactone) (PLCL)组成，其内层空隙小，适合上皮细胞生长，外层poly(L-lactic acid) (PLLA)/poly(e-caprolactone) (PCL)组成，其外层孔隙率大，适合平滑肌细胞及血管长入，我们制备的这种新型材料生物相容性好，力学强度大，在雄性新西兰兔2cm尿道缺损模型中试验，能够有效修复尿道缺损，在跟传统材料SIS，比较中，能够有更多尿路上皮，更多的血管长入材料。我们预计这种材料能够能够应用于尿道，以及其他中空组织的修复中去。