| 全仁夫.毛囊干细胞体外培养体系的建立*[J].浙江中西医结合杂志,2014,24(10): |
| 毛囊干细胞体外培养体系的建立* |
| The Experimental Study on Hair follicle stem cells in vitro system to establish QUAN Ren-fu., Xu Shi-chaoNi Yue-ming, et al. Department of Orthopaedics, Xiaoshan Traditional Chinese Medical Hospital, XiaoShan 311200, China. Corresponding author: QUAN Ren-fu, Email: quanrenfu@126.com. |
| 投稿时间：2014-03-03 修订日期：2014-05-21 |
| DOI： |
| 中文关键词: 毛囊干细胞 IV型胶原 Q-PCR |
| 英文关键词:: hair follicle stem cells,type IV collagen,Q-PCR |
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| 中文摘要: |
| 【】 目的：建立一整套可行的毛囊干细胞体外培养体系,获得高纯度的种子细胞,为构建新型人工皮肤积累实验数据,从而更好地解决皮肤大面积缺损的临床难题。方法：1.采用显微分离技术结合中性蛋白酶、四型胶原酶分离单个毛囊组织,分别接种于没有包被的24孔板(A组),Matrigel基质胶包被的24孔板(B组)和成纤维细胞作为饲养层细胞的24孔板(C组),新型完全培养基培养细胞,然后采用IV型胶原差速贴壁法纯化毛囊干细胞,计算其粘附率,比较筛选先后细胞的增值能力,并对其进行免疫荧光检测和Q-PCR检测与分析。结果：通过显微切割技术结合中性蛋白酶和IV型胶原酶,分离单个毛囊组织,采用新型完全培养基,Matrigel基质胶包被原代细胞培养,IV型胶原差速贴壁法分选纯化出的细胞,经过免疫荧光、细胞形态观察、Q-PCR检测,证实是毛囊干细胞,且增值力强,可大量扩增。结论：建立了一整套相对比较成熟的体外培养体系,为后期转染毛囊干细胞,构建复合人工皮肤,进行动物实验提供了充实的实验依据, 为新型人工皮肤的临床应用提供科学参考依据。 |
| 英文摘要: |
| Objective:To establish a set of viable hair follicle stem cells in vitro system, and get high purity of the hair follicle stem cells,It will provide substantial experimental evidence for building new composite artificial skin,and to slove the clinical problem of the treatment for the large areas of skin defect betterly. Methods: 1. Rat single hair follicles are separated through microscopy techniques combined with neutral protease,IV collagen enzyme in vitro,then,the single hair follicle tissues were seeded in no-coated 24-well plates (group A),matrigel package 24-well plates (group B) and mouse fibroblasts as feeder layer cells in 24-well plates(group C),and culture cells by new complete medium,and sublimate the hair follicle stem cells on basis of adhering to type IV collagen,calculate the adhesion rate,and contrast the cells" identification and value-added ability before and after the screening,last to do immunofluorescence detection and Q-PCR detection and analysis. Results: The cells which are obtained by separation of a single hair follicle tissue through microscopy techniques combined with neutral protease,IV collagen enzyme in vitro,and use the new complete medium,Matrigel-coated primary cell culture,and sublimate the cells on basis of adhering to type IV collagen are hair follicle stem cells after immunofluorescence, cell morphology, Q-PCR test confirmed , and has the strong proliferation ability. Conclusion: A set of relatively mature vitro culture system is estabished.It will provide substantial experimental evidence for the subsequent experiment of transfecting hair follicle stem cells, and building new composite artificial skin,and lay the foundation for hair follicle stem cells on the application of tissue engineering.And then it will provide scientific reference for the clinical application of the new artificial skin. |
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