中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (50): 9335-9338.doi: 10.3969/j.issn.1673-8225.2011.50.007

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

改良型混合酶消化法培养小鼠乳腺上皮细胞

易  琼1，王  鲁1，李圆方2，朱  伟2   

  1. 1贵州大学动物科学学院，贵州省贵阳市  550025
    2 贵州省生化工程中心，贵州省贵阳市  550025
  • 收稿日期:2011-06-28 修回日期:2011-10-26 出版日期:2011-12-10 发布日期:2011-12-10
  • 通讯作者: 王鲁，教授，博士，硕士生导师。贵州大学动物科学学院，贵州省贵阳市 550025 wanglu7007@ 163.com
  • 作者简介:易琼★，女，1987年生，湖南省株洲市人，汉族，贵州大学在读硕士，主要从事动物药理学研究。 yiqiong19870328@126.com
  • 基金资助:

    国家自然科学基金(31060347)；贵州大学研究生创新基金(校研农2010020)。

Cultivation of mouse mammary epithelial cells by using improved collagenase/trypsin digestion method

Yi Qiong1, Wang Lu1, Li Yuan-fang2, Zhu Wei2   

  1. 1Animal Science College of Guizhou University, Guiyang  550025, Guizhou Province, China
    2Center of Biochemical Engineering in Guizhou Province, Guiyang  550025, Guizhou Province, China
  • Received:2011-06-28 Revised:2011-10-26 Online:2011-12-10 Published:2011-12-10
  • Contact: Wang Lu, Doctor, Professor, Master’s supervisor, Animal Science College of Guizhou University, Guiyang 550025, Guizhou Province, China wanglu7007@163.com
  • About author:Yi Qiong★, Studying for master’s degree, Animal Science College of Guizhou University, Guiyang 550025, Guizhou Province, China yiqiong19870328@126.com
  • Supported by:

    the National Nature Science Foundation of China, No. 31060347*; Innovation Foundation for Graduate in Guizhou University, No. Xiaoyannong 2010020*

PDF

462

被引次数

15

摘要：

背景：目前采用的A型胶原酶和胰蛋白酶混合酶分离乳腺细胞团的方法操作复杂，实验条件要求高。
目的：观察改良型混合酶消化法能否成功进行乳腺上皮细胞的体外培养。
方法：采用Ⅰ型、Ⅱ型胶原酶和胰蛋白酶(1∶1∶1)混合消化直接用眼科剪剪碎的小鼠乳腺组织，37 ℃振荡消化获取乳腺细胞团，并采用差速贴壁法去除成纤维细胞，将细胞团接种于细胞培养瓶进行培养。
结果与结论：倒置显微镜下观察显示，改良型混合酶消化法能获得较多的细胞团，且培养12 h后细胞团绝大多数贴在细胞瓶壁，培养72 h后细胞团完全铺开融合成片，细胞呈典型的铺路石样。免疫荧光细胞化学染色结果显示，培养细胞角蛋白18呈阳性反应。说明应用改良型混合酶消化法能在短时间内获得大量较纯的乳腺上皮细胞。

关键词: 改良型混合酶消化法, 乳腺上皮细胞, 角蛋白18, 细胞培养, 小鼠

Abstract:

BACKGROUND: The mixture of collagenase A and trypsin that used to isolate the mammary epithelial cells is complex and strict to operate.
OBJECTIVE: To clarify whether mammary epithelial cells can be successively cultured with improved collagenase/trypsin digestion method in vitro.
METHODS: The mixture of typeⅠand Ⅱcollagenase and trypsin (1:1:1) was used to digest breast tissue, which was shredded directly with ophthalmic scissors. The organoids were obtained by 37 ℃ oscillation extraction and the differential adhesion method was used to remove the fibroblasts. Then the organoids were inoculated in the plastic dish.
RESULTS AND CONCLUSION: Inverted microscope observation showed that epithelial organoids could be mostly acquired and had attached to the plastic dish after 12 hours, and then cells were spreading out from them and forming confluent monolayer of cobblestone after cultured for 72 hours. In addition, the tissue-specific expression of cytokeratin 18 in mammary epithelial cells was identified by cytokeratin immunohistochemistry. It indicated that plenty of pure mammary epithelial cells could be harvested by using improved collagenase/trypsin digestion.

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