中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (53): 9965-9969.doi: 10.3969/j.issn.2095-4344.2012.53.017

• 细胞与组织移植 cell and tissue transplantation • 上一篇    下一篇

小鼠子宫内膜基质细胞分离培养、鉴定及体外蜕膜化的诱导

张 鹏1，钟 婷1，唐 珉2，张保平1，况海斌1   

  1. 南昌大学医学院 1生理学教研室， 2南昌大学医学院细胞生物学教研室，江西省南昌市 330006
  • 收稿日期:2012-04-12 修回日期:2012-06-25 出版日期:2012-12-30 发布日期:2012-12-30
  • 通讯作者: 况海斌，博士，教授，硕士生导师，南昌大学医学院生理学教研室，江西省南昌市 330006 khb9909@yahoo.com.cn
  • 作者简介:张鹏★，男，1986年生，湖北省孝感市人，汉族，南昌大学医学院在读硕士，主要从事生殖与内分泌研究。 zhangpeng10086@yahoo.cn

Mouse endometrial stromal cells: Isolation, culture, identification and induced decidualization in vitro

Zhang Peng1, Zhong Ting1, Tang Min2, Zhang Bao-ping1, Kuang Hai-bin1   

  1. 1Department of Physiology, School of Medicine, Nanchang University, Nanchang 330006, Jiangxi Province, China
    2Department of Cell Biology, School of Medicine, Nanchang University, Nanchang 330006, Jiangxi Province, China
  • Received:2012-04-12 Revised:2012-06-25 Online:2012-12-30 Published:2012-12-30
  • Contact: Kuang Hai-bin, Doctor, Professor, Master’s supervisor, Department of Physiology, School of Medicine, Nanchang University, Nanchang 330006, Jiangxi Province, China Khb9909@yahoo.com.cn
  • About author:Zhang Peng★, Studying for master’s degree, Department of Physiology, School of Medicine, Nanchang University, Nanchang 330006, Jiangxi Province, China zhangpeng10086@yahoo.cn

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1320

被引次数

10

摘要：

背景：国内已建立许多有关人的子宫内膜基质细胞分离培养及蜕膜化的诱导方法，但人的子宫标本存在取材困难等问题。
目的：建立小鼠子宫内膜基质细胞的分离培养及体外蜕膜化的诱导方法，为进一步研究子宫内膜蜕膜化的机制提供一种简单、有效的模型。
方法：通过胰酶与胶原酶Ⅱ消化及差时贴壁等方法，分离和纯化小鼠妊娠第4天的子宫内膜基质细胞；用细胞免疫化学方法鉴定基质细胞；通过孕酮和雌二醇联合处理诱导子宫内膜基质细胞的蜕膜化，然后用苏木精染核、实时定量PCR和免疫印迹方法检测蜕膜化标志分子的变化。
结果与结论：①分离培养的子宫内膜基质细胞的存活率为(90.1±2.3)%。②细胞免疫化学方法染色显示，分离纯化的子宫基质细胞Vimentin染色呈阳性，而Cytokeratin染色呈阴性，细胞纯度可达(92.3±2.4)%。③孕酮和雌二醇处理48，72 h后，苏木精染色显示细胞呈现多核化，蜕膜化标志分子dPRP、周期蛋白D3、孕酮受体mRNA随着培养时间延长均明显增加(P < 0.05)。结果可见采用酶消化和差时贴壁方法，可获得高纯度的子宫内膜基质细胞；子宫内膜基质细胞在体外通过孕酮和雌二醇联合诱导可产生蜕膜化。

关键词: 子宫, 基质细胞, 蜕膜化, 孕酮, 雌二醇, 蜕膜化催乳素相关蛋白, 周期蛋白D3, 孕酮受体, 组织构建

Abstract:

BACKGROUND: A lot of methods about the isolation, culture and induced decidualization in vitro of endometrial stromal cells have been established in China, but there is a problem about drawn difficulties of uterine specimens.
OBJECTIVE: To establish the method of isolation, culture and induced decidualization in vitro of mouse endometrial stromal cells, and to provide a simple and effective model for further study about the mechanism of endometrial decidualization.
METHODS: The trypsin and collagenase Ⅱ digestion and pasted wall were used to isolate and purify endometrial stromal cells of the mouse that pregnancy for 4 days; stromal cells were identified by immunocytochemistry; endometrial stromal cells treated by estradiol and progesterone were induced the decidualization, and the decidual markers were checked by hematoxylin nuclear staining, real-time quantitative PCR and western blot.
RESULTS AND CONCLUSION: The survival rate of endometrial stromal cells was (90.1± 2.3)%. Immunocytochemistry staining results showed that separated and purified uterine stromal cells were stained positive with Vimentin, and negative with Cytokeratin, the purities of cells were (92.3±2.4)%. Hematoxylin staining showed that endometrial stromal cells were multinucleated after treated with estradiol and progesterone for 48 and 72 hours. The decidualization prolactin-related protein, cyclin D3, progesterone receptor mRNA of decidual markers were significantly increased with the incubation time prolonged (P < 0.05). Highly purified stromal cells of endometrium can be obtained by enzymolysis and pasted wall purification. Endometrial stromal cells treated by estradiol and progesterone can be induced to the decidualization in vitro.

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