[1]孙小闵,伍津津,唐书谦,等.人皮肤成纤维细胞的搅拌及移行培养的初步研究[J].第三军医大学学报,2010,32(05):446-449.
 Sun Xiaomin,Wu Jinjin,Tang Shuqian,et al.Stirring-migration culture for human dermal fibroblasts and biological features[J].J Third Mil Med Univ,2010,32(05):446-449.
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人皮肤成纤维细胞的搅拌及移行培养的初步研究(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
32卷
期数:
2010年第05期
页码:
446-449
栏目:
论著
出版日期:
2010-03-15

文章信息/Info

Title:
Stirring-migration culture for human dermal fibroblasts and biological features
作者:
孙小闵伍津津唐书谦杨亚东杨桂红杨涛
第三军医大学大坪医院野战外科研究所皮肤科
Author(s):
Sun Xiaomin Wu Jinjin Tang Shuqian Yang Yadong Yang Guihong Yang Tao
Department of Dermatology, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, 400042, China
关键词:
成纤维细胞皮肤细胞培养技术细胞运动细胞增殖细胞形状人类
Keywords:
fibroblasts skin cell culture techniques cell movement cell prolife ration cell shape human
分类号:
R322.99;R329-33;R329.24
文献标志码:
A
摘要:
目的  建立人皮肤成纤维细胞(human dermal fibroblasts,HDFs)的简易三维搅拌、移行培养系统,鉴定所培养的HDFs生物学特性。  方法  酶消化包皮后进行原代、传代培养,选取第5~8代HDFs进行搅拌培养,设置静态培养对照组。倒置相差显微镜和扫描电镜进行HDFs在微载体上的生长情况、细胞形态以及后续移行培养的观察,免疫组织化学法进行细胞抗原表型的鉴定,ELISA法间接检测移行培养液中Ⅰ型胶原的含量。  结果  HDFs在微载体上的贴壁速度快,贴壁状况好,镜下观察细胞形态与静态培养均无差异。移行培养过程中HDFs能从微载体上移行到培养瓶底面进而较快增殖,呈梭形或多角形的正常形态和放射状、漩涡状贴壁生长,Ⅰ型胶原蛋白含量的间接测定呈现与细胞量变化相应的趋势均表明该体系培养的HDFs具有正常的生物学合成和分泌功能。  结论  成功建立的HDFs简易搅拌、移行培养系统是一种能进行活体外快速扩增细胞的培养方法,培养的细胞形态和功能良好,能满足组织工程皮肤实验和生产各阶段对种子细胞的需求。
Abstract:
Objective  To establish a simple stirring-migration culture system for human dermal fibroblasts (HDFs) by stirring in the three-dimensional cultivation monitored by electron microscope and to research its migration primarily.   Methods  Skin-derived primary HDFs were obtained by enzymatic digestion of foreskin, and then the cells at passage 5 to 8 were further cultured in the presence of microbeads with stirring for in vitro amplification. Static culture was also set up as control. The morphology of HDFs on the microcarriers in the stirring culture system was observed with inverted phase contrast microscopy and scanning electron microscopy (SEM). Then the cells were migrated from the attached microcarriers to tissue culture flasks. The cells were observed in every 24 h by inverted phase contrast microscopy. HDFs phenotype was identified with immunocytochemical staining against collagen Ⅰ and vimentin, and ELISA for the content of collagenⅠ in the supernatant of migration culture indirectly.   Results  HDFs adhered to microcarriers quickly with normal appearance. The morphology of HDFs did not show significant changes when compared with HDFs cultured in normal static culture. HDFs automatically and quickly migrated from microcarriers to the bottom surface of culture flask firmly. HDFs showed fusiform or polygonal appearance and their growth patterns showed blossom or whirlpool. So the morphology and growth pattern of these migrated cells were normal, in addition to the expression of collagen I and vimentin, and collagen Ⅰ secretion in accordance with the cells’number.   Conclusion  The established stirringmigration culture of HDFs is a simple method for quick amplification of HDFs in vitro, which may meet the demand for various experiments or production of seed cells in tissue engineering.

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更新日期/Last Update: 2010-03-05