上海交通大学学报（医学版）

›› 2012, Vol. 32 ›› Issue (7): 955-.doi: 10.3969/j.issn.1674-8115.2012.07.029

• 技术与方法 • 上一篇    下一篇

可用于基因表达研究的转基因小鼠乳腺上皮细胞模型的构建

蒋世忠1,2, 闫亚彬1,2, 谢 飞1,2, 王 娟1,2,3, 黄 英1,2,3, 任兆瑞1,2,3   

  1. 1.上海市儿童医院上海医学遗传研究所, 上海 200040； 2.上海交通大学医学遗传研究所, 上海 200040； 3.上海市胚胎与生殖工程重点实验室暨卫生部医学胚胎分子生物学重点实验室, 上海 200040
  • 出版日期:2012-07-28 发布日期:2012-08-17
  • 通讯作者: 任兆瑞, 电子信箱: zhrren@sjtu.edu.cn。
  • 作者简介:蒋世忠（1967—）, 男, 副主任医师, 博士；电子信箱: doctorjiang1517@sina.com。
  • 基金资助:

    国家科学与技术重大专项(2009ZX08007-003B)

Model establishment of transgenic mouse mammary epithelial cells for research of gene expression

JIANG Shi-zhong1,2, YAN Ya-bin1,2, XIE Fei1,2, WANG Juan1,2,3, HUANG Ying1,2,3, REN Zhao-rui1,2,3   

  1. 1.Shanghai Institute of Medical Genetics, Shanghai Children´s Hospital, Shanghai 200040, China； 2.Shanghai Institute of Medical Genetics, Shanghai Jiaotong University, Shanghai 200040, China； 3.Shanghai Key Laboratory of Embryo &|Production Engineering, Key Laboratory of Embryonic Molecular Biology, the Ministry of Health of China, Shanghai 200040, China
  • Online:2012-07-28 Published:2012-08-17
  • Supported by:

    National Science and Technology Major Program of China, 2009ZX08007-003B

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摘要：

目的 建立转基因小鼠乳腺上皮细胞模型，为研究乳腺内环境因素调控外源基因的表达和制备高效表达外源基因的乳腺生物反应器提供研究平台。方法 通过机械破碎和胶原酶消化的方法获取人转铁蛋白（hTF）转基因小鼠的乳腺上皮细胞，并进行体外原代培养。经胰蛋白酶纯化后，绘制乳腺上皮细胞生长曲线；免疫组织化学方法检测角蛋白18的表达；透射电子显微镜（透射电镜）观察乳腺上皮细胞超微结构；流式细胞仪检测细胞周期分布；显微镜下分析乳腺上皮细胞核型。将牛催乳素真核表达载体（pCMVbPRL）转染至转基因乳腺上皮细胞中，检测外源bPRL的表达。结果 乳腺上皮细胞生长曲线呈“S”形；免疫荧光组织化学分析结果显示hTF转基因小鼠乳腺上皮细胞角蛋白18呈阳性表达；透射电镜观察发现乳腺上皮细胞胞核较大、偏位，有双核或多核，胞质丰富，空泡、粗面内质网、高尔基体丰富；流式细胞仪检测结果显示：乳腺上皮细胞增殖活跃，G2/M期+S期细胞占15%；细胞核型分析结果显示处于分裂期的细胞具有正常的二倍体，染色体完整。pCMV-bPRL转染乳腺上皮细胞24 h后，上清液中检测到bPRL的表达。结论 体外培养的转基因乳腺上皮细胞具有类似体内细胞的生物学特征，并可表达真核表达载体，为研究环境因素对乳腺功能的影响及制备乳腺生物反应器提供了一种细胞模型。

关键词: 转基因小鼠, 乳腺上皮细胞, 真核表达载体, 牛催乳素

Abstract:

Objective To establish a cell model of transgenic mouse mammary epithelial cells for the research of foreign gene expression regulated by environment factors in the mammary gland as well as for the construction of mammary gland bioreactor. Methods The mammary epithelial cells of human transferrin (hTF) transgenic mouse were obtained by mechanical disruption and collagenase digestion, and followed by primary culture. After purification by trypsase, the growth curve of mammary epithelial cells was drafted. The expression of keratin 18 was detected by immunohistochemistry, the ultrastructure of mammary epithelial cells was observed by transmission electron microscopy, the distribution of cell cycles was determined by flow cytometry, and the karyotype of mammary epithelial cells was analysed under microscope. Eukaryotic expression vectors of bovine prolactin (pCMV-bPRL) were transfected into transgenic mammary epithelial cells, and the expression of exogenous bPRL was detected. Results The growth curve of mammary epithelial cells exhibited “S” shape. Immunofluorescence analysis revealed that there was positive expression of keratin 18 in hTF transgenic mouse mammary epithelial cells. Transmission electron microscopy demonstrated that the nuclei were bigger in mammary epithelial cells, with abundant cytoplasm, vacuole, rough endoplasmic reticulum and golgi body. Flow cytometry indicated that the proliferation of mammary epithelial cells was active, and cells in G2/M phase or S phase accounted for 15%. Cells in division stages had normal diploid and intact chromosome. Twenty-four hours after transfection of mammary epithelial cells by pCMV-bPRL, the expression of bPRL was detected in the supernatant. Conclusion The transgenic mouse mammary epithelial cells cultured in vitro have in vivo biological characteristics and capacity of expression of eukaryotic gene vectors, which may provide a good cell model for investigation of environment factors on the function of mammary gland and generation of mammary gland bioreactor.

Key words: transgenic mouse, mammary epithelial cell, eukaryotic gene vector, bovine prolactin