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
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摘要
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)×104times and(6.61±3.62)×103times, 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.
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)×104times and(6.61±3.62)×103times, 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.
引文
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[12]梁爽,李权,唐休发,等.静电纺丝纳米纤维膜作为骨骼肌组织工程支架材料的细胞相容性[J].中国组织工程研究与临床康复,2011,15(12):2171-2174.
[13]胡玮,崔新爱,刘欣,等.胶原/丝素电纺纳米纤维膜体外培养肝细胞的研究[J].中国生物医学工程学报2012,31(4):582-587.
[14]高小寒,魏蓓蓓,田玲玲,等.乳液法静电纺丝PLGA组织工程缓释纤维的研究[J].现代生物医学进展,2011,11(12):2224-2226.
[15]尹桂波,张琴.静电纺丝素/BMP-2支架在骨组织工程中的应用研究[J].国外丝绸,2007(3):12-15.
[2]杨青,李秀兰,张杨,等.表皮干细胞与毛囊干细胞生物学特性的比较[J].中国组织工程研究,2012,16(27):5034-5038.
[3]范微微,吕伟,范巨峰.人正常泌尿系上皮细胞系作为组织工程化尿道种子细胞的可行性研究[J].中国美容医学,2012,21(3):433-435.
[4]Li C,Xu YM,Song LJ,et al.Urethral reconstruction using oral keratinocyte seeded bladder acellular matrix grafts[J].J Urol,2008,180(4):1538-1542.
[5]Fu Q,Deng CL,Song XF,et al.Long-term study of male rabbit urethral mucosa reconstruction using epidermal cell[J].Asian J Androl,2008,10(5):719-722.
[6]王祝迁,木拉提·热夏提,李佳,等.大鼠触须毛囊干细胞的分离培养和鉴定[J].中国组织工程研究与临床康复,2011,15(27):5031-5034.
[7]杨震,顾建英,亓发芝.人体毛囊单位移植裸鼠背部创面效果的研究[J].中国临床医学,2011,8(6):764-767.
[8]张群,杨光辉,丛笑倩,等.毛囊干细胞表皮膜片修复裸鼠皮肤缺损的实验研究[J].组织工程与重建外科,2009,5(2):79-82.
[9]彭文标,刘春晓,谢群,等.富集兔毛囊干细胞构建组织工程化尿道的研究[J].上海交通大学学报(医学版)2013,33(3):285-289.
[10]傅强,邓辰亮,殷德明,等.脱细胞膀胱粘膜下筋膜修复尿道的实验研究[J].中国男科学杂志,2006,20(3):18-22.
[11]韩平,宋超,杨宇如,等.尿道脱细胞基质修复兔尿道缺损的实验研究[J].南方医科大学学报,2009,29(1):124-127,132.
[12]梁爽,李权,唐休发,等.静电纺丝纳米纤维膜作为骨骼肌组织工程支架材料的细胞相容性[J].中国组织工程研究与临床康复,2011,15(12):2171-2174.
[13]胡玮,崔新爱,刘欣,等.胶原/丝素电纺纳米纤维膜体外培养肝细胞的研究[J].中国生物医学工程学报2012,31(4):582-587.
[14]高小寒,魏蓓蓓,田玲玲,等.乳液法静电纺丝PLGA组织工程缓释纤维的研究[J].现代生物医学进展,2011,11(12):2224-2226.
[15]尹桂波,张琴.静电纺丝素/BMP-2支架在骨组织工程中的应用研究[J].国外丝绸,2007(3):12-15.