尿道移行上皮细胞与生物可降解尿道支架体外复合培养的研究

目的：研究体外培养的兔尿道上皮细胞在生物可降解性网状尿道支架上的贴附和生长增殖情况，观察其对尿道上皮细胞形态和功能的影响，利用组织工程技术培养种植细胞的尿道内支架。方法：应用机械分离与酶消化法分离培养兔尿道移行上皮细胞，并在体外行原代培养与扩增后制成细胞悬液接种在网状尿道支架上，形成尿道移行上皮细胞-支架复合物。采用免疫组织化学、荧光染色法鉴定尿道上皮细胞及其活性；采用倒置显微镜、扫描电镜观察尿道上皮细胞在支架表面吸附与生长状态。结果：网状尿道支架具有良好的生物相容性，能使尿道移行上皮细胞增殖，不影响其活性。尿道移行上皮细胞在尿道支架上贴附生长良好，1～2天后完全贴壁，3～7天细胞生长增殖活跃，支架网眼内充满上皮细胞，长期培养仍保持尿道移行上皮细胞特性，扫描电镜可见上皮细胞与网状支架贴附紧密，适度伸展并有基质分泌。结论：网状尿道支架适合尿道移行上皮细胞黏附生长．可作为尿道组织工程的细胞载体，利用组织工程方法可获得适于移植尿道细胞的组织工程化尿道。

Study on Iimplantation of Urethral Transitional Epithelial Cells on Artificial Biodegradable Urethral Scaffold IN Vitro

Objective：To study the adhesion and growth ability of rabbit urethral transitional epithelial cells on a three dimensional biodegradable polymer （PLLA） scaffold, to observe the influence of the morphology and rune tion of the urinary tract epithelial cells, and to reconstruct rabbit urethral scaffold by tissue engineering. Methods： Rabbit urethral transitional epithelial cells were isolated by mechanical and enzyme digested methods from adult rabbit urethra resected in urethrectomy and were seeded onto PLLA scaffold after expansion by in vitro culture. The complex of cell scaffold were then cultured ex-vivo. The growth and function of urethral ceil were observed and measured with immunohistochemical analyses during the cell seeded scaffolds in different culture intervals. The inverted microscopy and scanning electron microscopy were utilized to verify the interactions between the cells and the biomaterial. Results： Rabbit urethral transitional epithelial cells were well distributed and adhered to PLLA scaffolds and maintained their characteristics throughout the culture period. The seeded urethral cells could adherence to the scaffolds for 1- 2 d. After cultured in vivo for 3-7 d the ceil seeded scaffolds grew like tissues. Scanning electronic microscopy showed that cells clung to the silk scaffold tightly and there were a lot of extra cellular matrix surrounding cells. Immuocytochemical studies revealed that the cells were stained positively for cytokeratin in experimental group. Conclusions：PLLA scaffolds can support the urethral epithelial cells＇proliferation, and can be suitable carrier for tissue engineering of artificial urethra. It is feasibility that reconstruction of urethral tissue using tissue engineering technique.