输尿管种子细胞分离及组织工程输尿管网状支架构建

Isolation of ureter epithelial cells and construction of tissue engineering ureteral mesh scaffold

目的探讨输尿管上皮种子细胞的分离培养和增殖规律，以及构建种植种子细胞输尿管组织工程网状支架的可行性。方法采用自制输尿管翻转分离器，分离实验用小型猪输尿管上皮种子细胞。体外培养并鉴定输尿管上皮种子细胞。观察输尿管种子细胞形态变化，噻唑蓝（MTT）比色法检测原代、第3代、第5代和第7代种子细胞增殖特性。后将输尿管种子细胞种植到电纺丝输尿管网状支架，于支架孵育第1、3、5、7、9天检测种子细胞增殖活性，并绘制细胞生长曲线。结果成功分离培养输尿管上皮种子细胞，角蛋白抗体和尿路上皮特异抗体UroplakinⅡ证实为输尿管尿路上皮细胞。输尿管上皮种子细胞接种24h基本贴壁，呈多角形团簇样生长，8d时细胞融合70％以上，呈铺路石状排列。输尿管上皮种子细胞在电纺丝输尿管网状支架上生长增殖良好。结论改良输尿管上皮细胞分离方法可获得大量纯化的输尿管上皮种子细胞，其在输尿管网状支架上生长增殖良好，成功构建输尿管组织工程网状支架有望应用于输尿管损伤替代修复。

Objective To investigate the isolation and proliferation of ureter epithelial cells, and the feasibility of construction of tissue engineering ureter. Methods The ureters of minipigs were excised under sterile conditions. Homemade ureteral cell dissection tools were made and used to turn the ureteral mucosal surface outside. Ureteral epithelial cells were isolated and resuspended in Defined Keratinocyte SFM. Immunohistochemistry was used to identify cells. Morphology, growth and proliferation of the primary ureteral epithelial cells and cells from passage 3, 5 and 7 were observed. Cells were then seeded onto an eleetropspun polylactie acid scaffold to investigate the feasibility of construction of tissue engineering ureter. At the 1st, 3rd, 5th, 7th and 9th day of incubation, MTT assay was adopted to get a cell growth curve. Resuits Immunohistoehemistry and morphology demonstrated that the cultured cells were exclusively ureteral epithelial cells and retained the properties of normal urothelium. Most of the cells attached to the culture dish and began to spread after 24 h. The cultured ureteral epithelial cells possessed a typical appearance of cobblestone and their confluence reached up to 70% at 8th day. The seeded cells on the scaffold grew well and had a similar growth curve as cells growing on a culture dish. Conclusion The method of isolation can harvest a larger number of homogeneous ureteral epithelial cells. The cells can be used as seed cells for constructing tissue engineering ureter which can be used for reconstruction of injured ureter.