富集兔毛囊干细胞构建组织工程化尿道的研究

Study on constructing tissue engineered urethra by enriched rabbit hair follicle stem cells

目的富集兔毛囊干细胞构建组织工程化尿道,观察其对尿道缺损的修复效果。方法应用机械分离与酶消化法分离培养兔毛囊细胞,体外原代培养扩增至3×106个,采用流式细胞仪富集毛囊干细胞,分选直径<10μm的integrin-α6+/cd71-细胞,免疫荧光法检测细胞K19、p63和β1整合素表达。将毛囊干细胞接种于膀胱脱细胞基质支架材料上,构建组织工程化尿道,扫描电子显微镜观察细胞在支架上的分布状态。利用组织工程化尿道修复2.5 cm的兔尿道缺损,经尿道造影和组织学检查观察缺损修复效果。结果免疫荧光法检测结果显示毛囊干细胞K19、p63、β1整合素表达阳性。扫描电子显微镜观察发现细胞与支架紧密贴附,细胞伸展适度且有基质分泌。组织学检查和免疫组织化学检测发现,毛囊干细胞形成复层上皮结构,AE1/AE3染色阳性。组织工程化尿道修复缺损后3个月,尿道造影显示尿道平滑通畅。结论通过调整优化分选参数富集兔毛囊干细胞,构建的组织工程化尿道可用于修复兔尿道缺损。

Objective To construct tissue engineered urethra with enriched rabbit hair follicle stem cells, and observe its effect in repair of urethral defect. Methods Rabbit hair follicle cells were isolated and cultured by mechanical separation and enzyme digestion, and were in vitro cultured and proliferated to 3×10^6. Integrin-α6＋/cd71 - cells of 〈 10μm in diameter were sorted by flow cytometry, and the expression of K19, p63 and β1 integrin was detected by immunofluorescence method. Hair follicle stem cells were seeded on bladder acellular matrix scaffold, tissue engineered urethra was constructed, and the cell distribution on scaffold was observed by scanning electron microscope. Rabbit urethral defect of 2.5 cm was repaired with tissue engineered urethra, and the effect of repair was observed by urethrography and histological examination. Results Immunofluorescence detection revealed that there was positive expression of K19, p63 and β1 integrin in hair follicle stem cells. Scanning electron microscope demonstrated that cells firmly attached to the scaffold and moderately stretched, and there was matrix secretion. Histological and immunohistochemical examinations indicated there was stratified epithelium-like structure, with positive staining of AE1/AE3 in hair follicle cells. Three months after repair of urethral defect by tissue engineered urethra, urethrography illustrated the urethra was smooth and unobstructed. Conclusion Tissue engineered urethra constructed by enriched rabbit hair follicle stem cells can be used for repair of rabbit urethral defect.