兔尿道上皮细胞的体外连续培养

Continuous culture of rabbit urethral epithelial cells in vitro

背景：尿道损伤后的瘢痕挛缩或缺损可导致狭窄，寻找理想的修复替代材料已成为研究热点。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。设计、时间及地点：单一样本观察，于2005—04／2007—02在解放军总医院泌尿外科完成。材料：健康成年雄性新西兰雄兔6只，体质量3．0～3．5kg：实验用DispaseⅡ为美国Gibco公司产品。方法：取雄性新西兰兔尿道黏膜组织，分离上皮采用DispaseⅡ工作液，上皮细胞间的分离采用混合消化酶（1．25g／L胰蛋白酶与0．2g／L乙二胺四乙酸等体积混合）消化，以差速贴壁法排除成纤维细胞。使用DMEM与F123：1混合培养液加体积分数为0．1的胎牛血清，对上皮细胞进行原代单纯培养和传代培养；取第2-6代细胞进行上皮细胞的免疫组织化学染色，以正常尿道黏膜组织石蜡切片为阳性对照组，以成纤维细胞铺片为阴性对照组。主要观察指标：①利用倒置相差显微镜动态观察细胞形态、生长、增殖情况。②采用活细胞荧光染色法观察细胞存活状态。③以扫描电镜观察细胞超微结构。④采用免疫组织化学染色行细胞鉴定。结果：①原代培养3-5d细胞逐渐生长融合成片，如铺路石状，细胞大小均一。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9～10代。②第4～6代细胞在免疫荧光染色和超微结构观察中显示良好形态。③免疫组织化学证实角蛋白染色阳性。结论：应用组织工程技术，成功培养尿道上皮种子细胞。结果表明，细胞生长状态良好，增殖较快。经特异性免疫组织化学及形态学鉴定为纯化的尿道上皮细胞。

BACKGROUND： Cicatricial contracture or defects may result in the stenosis after urethral injury, it is an urgent research topic to find out an ideal repair material. 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. DESIGN, TIME AND SETTING： A single sample observation was performed at the Department of Urinary Surgery in Chinese PLA General Hospital between April 2005 and February 2007. MATERIALS： Six healthy adult male New Zealand rabbits, weighing 3.0-3.5 kg, were adopted; Dispase II was produced by Gibco Company （USA）. METHODS： The urethral mucosa from male New Zealand rabbits was harvested, then epithelium and endothelial cells were isolated with Dispase Ⅱ and digested with mixed enzyme （1.25 g/L trypsinase and 0.2 g/L edetic acid）, and the fibroblast were removed by differential centrifugation. Primary culture and passage culture of the endothelial cells were conducted in the medium containing DMEM and F12 at the ratio of 3 ： 1, adding 0.1 volume fraction of fetal calf serum. The cultured cells at passages 2-6 were analyzed with immunohistochemistry. Paraffin section of normal urethral mucosa was taken as positive control group, and fibroblast slides were taken as negative control group. MAIN OUTCOME MEASURES： Morphology, growth and proliferation of cells were observed dynamically under inverted phase contrast microscope. Cell survival was determined by living cell fluorescence staining. The ultra-microstructure of cells was observed under scanning electron microscope. Cell identification was performed by immunohistochemistry. RESULTS： The primarily cultured cells fused when they had been cultured for 3-5 days. They were shaped as paving roads. During the growth phase, the cultured cells were all epithelial cells without fibroblasts, and could be subcultured 9-10 generations. Good configuration was found at passages 4-6 by immunofluorescence and electron microscopy.Immunohistochemistry revealed that keratin staining was positive. CONCLUSION： By means of tissue engineering techniques, the urethral epithelium seed cells can be cultured successfully, and maintain the ability to grew well and proliferate rapidly. They have been identified as the purified urethral epithelial cells by specific immunohistochemistry and morphology.