大鼠背部毛乳头细胞的分离培养和鉴定  

Cultivation and identification of follicular papilla cells from back skin of actual rat in vitro

作　　者：郑国玺[1] 朱珠[1] 祝康[1] 侯瑾[1] 韦俊荣[1] 夏翠[1] 许珉[1] 

机构地区：[1]西安交通大学医学院第二附属医院耳鼻咽喉科,西安710004

出　　处：《临床耳鼻咽喉头颈外科杂志》2012年第13期603-607,共5页Journal of Clinical Otorhinolaryngology Head And Neck Surgery

基　　金：国家自然科学基金资助项目(No:30471877);陕西省科技攻关项目(No:2010K15-08)

摘　　要：目的:建立一种高效分离成年SD大鼠背部毛乳头细胞(DPCs)的方法,考察DPCs在体外培养条件下的生长特性。方法:采用改良二步酶消化法分离大鼠背部DPCs,应用相差显微镜进行形态学观察,流式细胞技术检测细胞周期,细胞计数法绘制生长曲线,流式细胞技术和免疫细胞化学法检测细胞表面分子标志。结果:分离培养的DPCs多呈短梭形,融合前细胞呈现凝集性生长趋势;传代细胞呈多角形或短梭形,传代后第3天开始呈对数生长,第9天达增殖高峰。第1、3、5代的细胞增殖时间分别为68、52和36h。流式细胞仪检测第1、3、5代细胞周期,处于G0/G1期分别为(90.21±5.13)%、(81.23±1.85)%和(75.16±5.32)%,随着传代次数的增加,G0/G1期细胞比例逐渐下降,但仍大部分处于静止期;经免疫化学SABC法染色显示,α-SMA抗体表达阳性,CK抗体表达阴性,细胞表面分子表达CD44、CD90,但不表达CD34。结论:改良二步酶消化法能成功分离培养成年SD大鼠背部DPCs,体外培养的DPCs与干细胞的生物学特性相吻合,有望成为一种新的细胞替代疗法的种细胞来源。Objective：To establish more efficient method to isolate of dermal papilla cells（DPCs） from back skin of SD rats,and then to study the growth ability and characteristics of SD rat dermal papilla cells in vitro.Method：DPCs were separated from back skin of SD rats according to the modified method of two-step enzymatic digestion.The DPCs morphological observation under inverted microscope,the growth kinetics by cells number,the cell cycle analysis by flow cytometry analysis and determine the surface epitopes by immunohistochemical staining and flow cytometry analysis.Result：Cultured DPCs were similar to fibroblasts in appearance,but generally and periodically exhibited an aggregative growth pattern.The growth kinetics showed that the number of DPCs presented progressive increase in a logarithm mode in the first 3 days and entered into plateau after 9 days,P1,P3,P5 multiplication time was 68 h,52 h and 36 h,respectively.The flow cytometr-ical analysis showed that DPCs of P1,P3,P5 G_0/G_1 stage were（90.21±5.13）%,（81.23±1.85）% and（75.16±5.32）%,respectively.G_0/G_1 stage cells became less following passage subculture and elongation of culture time,but most of the DPCs stayed resting stage stilly.The cultivated dermal papilla cells expression of α-smooth muscle and CD44 on cell surface was positive,CK and CD34 were negative.Conclusion：DPCs can be separated by the modified method of two-step enzymatic digestion successfully.The cultivated dermal papilla cells vitro show the feature of stem cells and has important potentially as a new seed cell source for cell engineering.

关 键 词：毛乳头细胞 间充质干细胞 真皮 组织工程 

分 类 号：R764[医药卫生—耳鼻咽喉科]