纳米微囊基因修饰PLGA生物支架的组织工程技术重建尿道的实验研究

The key of urethral reconstruction with tissue engineering is to prepare the scaffold with good structure and function which could provide an ideal growth microenvironment for seed cells and inhibit the formation of urethral scar.The biodegradable scaffold（poly L-glutamic acid）decorated by nano microcapsuls could mimic the extracellular matrix in the greatest extent because it has a high porosity, a high permeability and excellent biocompatibility. The cell may adhere to grow on a scaffold surfaces. In the same time, the scaffold could increase the activity of the genes obviously. In our previous study in vitro, it showed that TIMP-1 significantly increased levels of transforming growth factor β1(TGFβ1), Collagen I, α-SMA to promote the formation of urethral scar . Our preliminary study also showed that the down regulation of TIMP-1 high expression by constructed siRNA interfering technology could prevent fibroblasts synthesis type I collagen and inhibition of scar formation. At same time, we found that TIMP-1 disappeared quickly owing to short half-life period in vivo. The TIMP-1cann’t be regulated effectively.In order to maintain TIMP-1 siRNA expressed without any decay ,We plan to adopt the biodegradable scaffold (PLGA) decorated by nano microcapsuls gene, oral mucosa cells and fibroblasts as seed cells, so that we can build the nano tissue engineering urethra which can effectively inhibit scar formation .This experimental study will explore the new strategy for urethral reconstruction.

构建具有良好结构与功能的支架，为种子细胞提供理想的生长微环境，同时有效抑制尿道瘢痕的形成，是组织工程尿道重建的关键。纳米微囊修饰的可降解生物材料支架（PLGA）具有高孔隙率和高渗透性，细胞可在其表面粘附生长，明显提高转载基因活性。我们前期体外细胞实验发现，TIMP-1可增加成纤维细胞内TGFβ1、Ⅰ型胶原蛋白、α-SMA等关键因子的表达，形成尿道瘢痕。而采用siRNA技术能够干扰TIMP-1高表达，减少胶原合成，防止瘢痕过度形成。同时我们发现TIMP-1在体内半衰期短而灭活快，无法有效调控。为能保持TIMP-1 siRNA无衰减表达，故本研究拟采用纳米微囊包裹siRNA干扰的TIMP-1基因，并以此修饰PLGA，以口腔黏膜细胞和成纤维细胞为种子细胞，使构建的纳米组织工程尿道可有效抑制瘢痕的形成，降低尿道狭窄的复发率。

先天性的尿道缺陷如尿道下裂、以及获得性的由创伤、炎症、肿瘤、医源性损伤等因素所导致的尿道狭窄，是泌尿外科常见的疾病之一。尿道狭窄治疗的难点之一在于其复发率较高，而其根源在于尿道组织纤维化、尿道瘢痕的形成，其而在于尿道修复重建的材料缺少。本研究拟构建具有良好结构与功能的组织工程复合支架，为种子细胞提供理想的生长微环境，同时有效抑制尿道瘢痕的形成。分离培养动物口腔上皮细胞与成纤维细胞为种子细胞，筛选出具有抑制瘢痕生成作用的TIMP-siRNA,使用纳米微囊对siRNA进行包裹实现对于基因的保护以及缓释作用，制备生物相容性良好的水凝胶支架，混合种子细胞以及纳米微囊，构建出用于组织工程尿道修复的复合支架。用上述的复合支架修补尿道狭窄动物模型的尿道缺损，术后1个月尿道造影显示尿道管腔无明显狭窄，局部无明显炎症反应，此复合支架具有促进组织愈合以及抑制瘢痕形成的作用，对于日后组织工程尿道修复在临床中的应用有一定参考价值。