毛囊干细胞结合电纺纳米丝素纤维构建组织工程尿道膜片的体外研究

Study of Constructing Tissue Engineered Urethra Membrane Using Hair Follicle Stem Cells and Nano Silk Fiber in Vitro

目的探索毛囊干细胞结合电纺纳米丝素纤维,构建组织工程尿道膜片的可行性。方法使用酶消化法分离兔毛囊细胞,利用流式细胞仪分选毛囊干细胞,体外原代培养扩增,计算细胞的最大扩增倍数及克隆形成率;使用流式细胞仪检测细胞K19、p63、CD29的阳性表达率。将毛囊干细胞接种在电纺纳米丝素纤维支架上,构建组织工程尿道。应用组织学、荧光染色法观察组织工程尿道的体外构建情况。结果兔毛囊干细胞与兔毛囊细胞的最大扩增倍数分别为(5.92±0.77)×104倍和(6.61±3.62)×103倍;兔毛囊干细胞与兔毛囊细胞的克隆形成率分别为(9.07±1.18)%和(3.95±1.01)%。K19、p63、CD29在毛囊干细胞中表达率分别为(89.36±6.69)%、(92.10±5.49)%和(89.98±6.89)%;在兔毛囊细胞中分别为(39.38±2.20)%、(40.78±2.61)%和(40.57±2.79)%;毛囊干细胞在支架上能形成复层上皮样结构,AE1/AE3染色阳性。结论兔毛囊干细胞结合电纺纳米丝素纤维,能成功构建组织工程尿道,可用于体内修复的进一步研究。

Objective To explore the feasibility of constructing tissue engineered urethra membrane using hair follicle stem cells and nano silk fiber. Methods Hair follicle stem cells were isolated by enzymatic digestion and fluorescent cell sorting, then cultured in vitro. The maximum amplification factor and colony forming rate were calculated; K19, p63, CD29 positive expression rate were tested by flow cytometry. The hair follicle stem cells were then seeded on electro-spun nano silk fibers to construct tissue engineered urethra. The construction of tissue engineered urethra in vitro was observed by histological observation and fluorescence staining. Results The maximum amplification factor of rabbit hair follicle stem cells and normal hair follicle cells were （5.92±0.77）×10^4 times and （6.61±3.62）×10^3 times, and the colony forming rate were （9.07±1.18）%and （3.95±1.01）%respectively. The expression rate of K19, p63, CD29 in hair follicle stem cells were （89.36±6.69）%, （92.10±5.49）%, （89.98±6.89）%, and in normal hair follicle cells were （39.38±2.20）%, （40.78±2.61）%, （40.57±2.79）%. Stratified epithelium-like structure were formed after hair follicle stem cells seeded on the nano silk fibers, and AE1/AE3 staining was positive. Conclusion Rabbit hair follicle stem cells combining with electro-spun nano silk fibers can successfully construct tissue engineered urethral, which can be used for the further study of in vivo repair.