中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (28): 5220-5223.doi: 10.3969/j.issn.1673-8225.2010.28.021

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

人早孕绒毛滋养层细胞原代培养：差异贴壁法与消化排除法联合应用的可行性

张小红1，李玉红1，许  倩1，任春丽2   

  1. 1 承德医学院，河北省承德市  067000；2 承德医学院附属医院，河北省承德市 067000
  • 出版日期:2010-07-09 发布日期:2010-07-09
  • 通讯作者: 李玉红，教授，博士，承德医学院，河北省承德市 067000 youngcheer2003@yahoo.com.cn
  • 作者简介:张小红★，女，1975年生，河北省承德市人，满族， 2009年华北煤炭医学院毕业，硕士，实验师，主要从事滋养细胞肿瘤的恶性侵袭的分子机制研究。 308697922@qq.com
  • 基金资助:

    2009年河北省教育厅科学研究计划项目；2005年河北省卫生厅医学科学研究重点课题；2001年承德市科技局河北省科学技术研究与发展计划项目；2008年河北省教育厅自然科学研究计划项目；2009年承德医学院科学研究计划项目。

Primary culture of human chorionic trophoblast cells by differential attachment in combination with digestion elimination methods

Zhang Xiao-hong1, Li Yu-hong1, Xu Qian1, Ren Chun-li2   

  1. 1 Chengde Medical College, Chengde   067000, Hebei Province, China; 2 Affiliated Hospital of Chengde Medical College, Chengde   067000, Hebei Province, China
  • Online:2010-07-09 Published:2010-07-09
  • Contact: Li Yu-hong, Doctor, Professor, Chengde Medical College, Chengde 067000, Hebei Province, China youngcheer2003@yahoo.com.cn
  • About author:Zhang Xiao-hong★, Master, Experimentalist, Chengde Medical College, Chengde 067000, Hebei Province, China 308697922@qq.com
  • Supported by:

    the Scientific Research and Development Program of Hebei Education Department in 2009*; the Medical Scientific Research Key Program of Hebei Health Department in 2005*; the Science and Technology Research and Development Program of Hebei Province, Chengde Science and Technology Bureau in 2001*; the Natural Science Foundation Program of Hebei Education Department in 2008*; the Scientific Research Program of Chengde Medical University in 2009*

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摘要：

背景：国内外有关人绒毛膜滋养层细胞的体外培养方法，大多步骤繁琐，细胞纯度低而且成本很高，不适于普通实验室推广应用。
目的：拟求建立一种人妊娠5~10周绒毛滋养层细胞简便有效的分离培养及鉴定方法。
方法：采用改进的胰酶消化法分离培养妊娠5~10周人绒毛滋养层细胞，加0.062 5%胰酶，37 ℃消化25~40 min；以差速贴壁法和消化排除法纯化细胞，倒置显微镜观察细胞形态，细胞免疫化学方法鉴定细胞来源和纯度。
结果与结论：倒置显微镜下原代培养滋养层细胞接种后见大量圆形细胞悬浮存在，1 h后可见部分细胞贴壁，24 h后70%~80%细胞贴壁，五六天细胞数量明显增多，细胞呈三角形、多边形平铺片状生长，核大卵圆形居中，部分细胞连接成片，部分细胞呈长梭形。七八天长满瓶壁的80%~90%可传代。9~10 d铺满培养瓶底部。细胞碎屑不贴壁。传代接种后1.5 h贴壁，迅速增长，三四天爬满瓶底，各代细胞形态基本一致。可见细胞为上皮样细胞形态，呈片状铺展生长。细胞角蛋白染色阳性,波形蛋白染色阴性细胞达70%~80%。采用低浓度胰酶进行长时间消化分离培养人早孕绒毛滋养层细胞，利用差异贴壁法和消化排除法以及反复换液法进行纯化，可简单、快捷地获得较高纯度的人早孕绒毛滋养层细胞。

关键词: 滋养层细胞, 细胞培养, 细胞纯化, 细胞鉴定, 绒毛组织

Abstract:

BACKGROUND: The methods for primary culture of human chorionic trophoblast cells in vitro are tedious. Moreover, the low cell purity and high cost limit their application in ordinary laboratory.
OBJECTIVE: To establish a simple, convenient and practical method for primary culture of human chorionic trophoblast cells in vitro.
METHODS: Modified trypsinization was used to isolate chorionic trophoblast cells from 5-10 weeks gestational women by 0.0625% trypsin at 37 ℃ for 25-40 minutes. The cells were puvified by differential attachment and digestion method. The inverted microscope was used to observe the shape of cells, and immunocytochemistry was used to identify the source and the purity of cells．
RESULTS AND CONCLUSION: The microscopic examination revealed that following cultures, a large number of round cells freely floated; some cells began to attach 1 hour later, and 70%-80% cells were attached by 24 hours; the number of cells significantly increased by 5-6 days, displaying triangle or polygon shape and patchy spreading growth; the nuclei were large, oval and centralized; some cells were long-fusiform shaped. The 80%-90% confluent cells were passaged by 7-8 days, and completely confluent by 9-10 days. Cell debris did not attached. The passage cells attached 1.5 hours after culture, rapidly grew, and became confluent by 3-4 days. The appearance of each passage cells was similar, displaying epithelioid cell appearance. The 70%-80% cells were  positive for cytokeratin and negative for vimentin. Low density trypsinization, differential attachment and digestion elimination methods are simple and easy to obtain high purity human chorionic trophoblast cells．

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