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目的:探索应用小肠黏膜下基质(small intestinal submucosa,SIS)复合口腔黏膜细胞(oral keratinocytes,OK)和转染金属蛋白酶抑制剂-1(tissue inhibitor of metalloproteinase-1,TIMP-1)siRNA的成纤维细胞(fibroblasts,FB)构建组织工程化尿道的可行性。方法:分离培养OK,FB,并用TIMP-1siRNA转染FB,检测转染前后FB分泌Ⅰ型胶原的改变。剥离24只雄性兔子腹侧尿道黏膜2.0cm×0.8cm,并将其平均分为3组,每组8只。第1组应用单纯SIS修复,第2组应用SIS复合OK修复,第3组应用SIS复合OK和转染TIMP-1siRNA的FB修复。术后1个月、6个月行逆行尿道造影检查和组织学分析评估三组尿道重建的效果。结果:转染TIMP-1siRNA的FB分泌Ⅰ型胶原明显减少。逆行尿道造影下,第1组兔子中有5只尿道管腔通畅,第2组有6只,第3组有7只。组织学上,第1组在1个月时形成了不完整的尿路上皮,在6个月时纤维化和炎症较明显。第2组和第3组在1个月时形成了完整的尿路上皮,在6个月时纤维化和炎症较轻,且第3组生长的尿路上皮、平滑肌和血管较第2组明显增多。结论:OK和转染TIMP-1siRNA的FB可作为尿道组织工程中的种子细胞用于尿道修复重建,OK和转染TIMP-1siRNA的FB可抑制尿道瘢痕的产生。
OBJECTIVE: To explore the effect of fibronectin on the expression of small intestinal submucosa (SIS) combined with oral keratinocytes (OK) and tissue inhibitor of metalloproteinase-1 (TIMP-1) Feasibility of constructing tissue engineered urethra with fibroblasts (FB). Methods: OK and FB were isolated and cultured. FB was transfected with TIMP-1 siRNA to detect the changes of type Ⅰ collagen secreted by FB before and after transfection. The ventral mucosa of 24 male rabbits were stripped 2.0 cm × 0.8 cm and divided equally into 3 groups with 8 in each group. Group 1 applied SIS repair alone, Group 2 applied SIS OK repair, and Group 3 SIS combined OK and transfection of TIMP-1 siRNA for FB repair. At 1 month and 6 months after operation, retrograde urethral angiography and histological analysis were performed to evaluate the effect of urethral reconstruction. Results: Type Ⅰ collagen secreted by FB transfected with TIMP-1 siRNA was significantly decreased. Under retrograde urethrography, there were 5 urethral patency in group 1 rabbits, 6 in group 2 and 7 in group 3. Histologically, group 1 formed incomplete urothelium at 1 month, with fibrosis and inflammation more pronounced at 6 months. Group 2 and group 3 formed intact urothelium at 1 month, with less fibrosis and inflammation at 6 months, and urinary epithelium, smooth muscle and blood vessels in group 3 were significantly more developed than those in group 2 increase. CONCLUSIONS: OK and FB transfected with TIMP-1 siRNA can be used as seed cells in urethral tissue engineering for urethral repair and reconstruction. OK and FB transfected with TIMP-1 siRNA can inhibit urethral scarring.