种植自体尿路上皮细胞的包膜化输尿管组织工程支架的构建

Construction of a capsular tissue-engineered ureteral stent seeded with autologous urothelial cells

目的探讨应用组织工程技术体外构建种植自体尿路上皮细胞的包膜化聚乳酸输尿管支架的可行性。方法将聚乳酸输尿管支架于比格犬皮下包埋3周,在其表面诱导形成一层结缔组织薄膜,获得包膜化聚乳酸输尿管支架。经脱细胞处理后,将体外扩增培养的自体尿路上皮细胞种植于包膜输尿管支架表面。采用常规HE染色组织切片、扫描电镜和免疫组化法对包膜输尿管支架及尿路上皮附着情况进行观察;应用MTT比色法比较尿路上皮细胞在包膜化聚乳酸输尿管支架组和管状小肠粘膜下基质组的生长差异。结果 HE染色及Ⅷ因子免疫组化染色结果显示结缔组织层内含有大量毛细血管,但无明显局部炎症反应;扫描电镜和免疫组化染色提示包膜含有丰富的胶原纤维构成、表面呈现三维立体结构,尿路上皮细胞能在支架包膜表面粘附,形成连续的上皮层;MTT检测显示包膜化聚乳酸输尿管支架组和管状小肠粘膜下基质组组吸光光度值比较无明显差异(P>0.05),说明尿路上皮细胞在包膜化输尿管支架上能连续增殖。结论包膜化聚乳酸输尿管支架适合尿路上皮细胞黏附和增殖,体外构建的包膜化组织工程输尿管有潜在的临床应用价值。

Objective To investigate the feasibility of constructing a capsular poly L-lactic acid （PLLA） ureteral stent seeded with autologous urothelial cells using tissue engineering methods. Methods The capsular ureteral stent was constructed by subcutaneously embedding PLLA ureteral stent in the back of beagles for 3 weeks to induce the formation of connective tissue on the surfaces. After decellularization of the stent, the expanded autologous urothelial cells were seeded on the stent. The surface structure and cell adhesion of the stent were observed using HE staining, scanning electron microscope （SEM） and immunocytochemichal staining. MTT assay was used to evaluate urothelial cell proliferation on the capsular PLLA ureteral stent and on circumferential small intestinal submucosa graft. Results HE staining and VIII factor immunohistochemistry revealed numerous capillaries in the connective tissue encapsulating the stent without obvious local inflammatory response. The results of SEM and immunocytochemichal staining showed that the capsule contained rich collagenic fibers forming three-dimensional structures, and the seeded autologous urothelial cells could adhere and well aligned on the surface. MTT assay showed normal growth of the cells on the stent as compared with the ceils grown on circumferential small intestinal submucosa graft. Conclusion The capsular PLLA ureteral stent allows adhesion and proliferation of autologous urothelial cells and shows a potential in applications of constructing tissue-engineered ureter.