以L929细胞为滋养层的尿道黏膜上皮细胞体外培养

目的 探索尿道黏膜上皮细胞体外培养的技术和方法，为进一步采用组织工程技术构建尿道黏膜组织奠定基础，并为尿道黏膜的生理、药理、毒理学及微生态研究提供实验模型。方法 取刚离乳的雄性新西兰幼兔尿道黏膜组织，分别以DispaseⅠ消化液和混合消化液消化成单细胞悬液，以差速贴壁法排除成纤维细胞，接种后以L929细胞为滋养层细胞进行培养，定期换液，细胞生长、增殖至80％～90％融合时传代。细胞进行常规HE染色、流式细胞仪检测，以扫描电镜、透射电镜观察其超微结构。再分别设立实验组(n=20)、阳性对照组(正常尿道黏膜组织石蜡切片，n=20)及阴性对照组(成纤维细胞铺片，n=20)行免疫组织化学染色。结果 原代培养10天左右细胞逐渐生长融合成片，如铺路石状，细胞大小均一。上皮细胞为二倍体细胞，生长期内均为单一的上皮细胞，无成纤维细胞混杂生长，细胞可传11～13代，成活50～60天。结论 新西兰幼兔尿道黏膜上皮细胞可在体外进行培养，在一定时间内保持增殖活力，为构建组织工程化尿道奠定了基础，且为尿道黏膜的体外研究提供了实验模型。

URETHRAL EPITHELIUM CULTURE BY USING L929 CELLS AS TROPHODERM IN VITRO

OBJECTIVE: To study the technique and method of urethral epithelium culture in vitro, so as to lay the groundwork for reconstructing a tissue engineering urethra and to provide an experimental model of urethral mucosa in physiological, pathological, toxicological and microbiological study. METHODS: The urethral mucosa from a young male New Zealand hare that had just been out of milk, was digested into single cell liquid with Dispase II and mixed enzyme, and the fibroblast were removed. After being seeded, the cells were cultured by using L929 cells as trophoderm. The medium was changed regularly and the cells were subcultured when they grew to mix together 80% to 90%. The cultured cells were analyzed with histochemistry, immunohistochemistry dyeing and flow cytometry examination. We observed the ultrastructure of cells with scanning electron microscope and transmission electron microscope. RESULTS: The primary cultured cells fused when they had been cultured for about ten days. They were the same in size like road rocks. The cultured cells were all epithelial cells without fibroblasts and were diploid cells. The cells could be subcultured 11-13 generations, and could survive 50-60 days. CONCLUSION: The urethral epithelium of young New Zealand hare can be cultured in vitro and maintain the ability to proliferate within a certain time. The study result not only sets a role in reconstructing a tissue engineering urethral mucosa, but also provides an experimental model for the research of urethral mucosa in vitro.