国际妇产科学杂志

国际妇产科学杂志 ›› 2017, Vol. 44 ›› Issue (1): 113-117.

• 论著 • 上一篇    下一篇

人子宫内膜异位症在位子宫内膜干细胞的分离和鉴定

张继雯   

  1. 天津中医药大学第二附属医院
  • 收稿日期:2016-09-19 修回日期:2016-11-30 出版日期:2017-02-15 发布日期:2017-03-28
  • 通讯作者: 张继雯 E-mail:wen721913@126.com
  • 作者简介:2016-12-18
  • 基金资助:
    中国博士后科学基金;国家自然基金

Isolation and Identification of Stromal Stem Cells from Eutopic Endometrium Functionional layer of Women with Endometriosis

Ji-wen ZHANG   

  • Received:2016-09-19 Revised:2016-11-30 Published:2017-02-15 Online:2017-03-28
  • Contact: Ji-wen ZHANG E-mail:wen721913@126.com

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摘要： 目的 从子宫内膜异位症患者在位子宫内膜中分离、培养子宫内膜干细胞，并鉴定其生物学特性，探索一种简便、高效的分离人子宫内膜异位症子宫内膜干细胞的方法。方法：从诊刮获取的子宫内膜异位症患者功能层子宫内膜中分离出子宫内膜间质细胞，克隆形成法从该细胞中分离出具有强克隆形成能力的克隆形成细胞，对比非克隆形成细胞以MTT法检测两种细胞的增殖能力，连续克隆形成实验检测其克隆形成能力，微球体形成实验检测其微球体形成能力，诱导分化实验检测克隆形成细胞定向分化能力，裸鼠异位病灶形成实验检测其异位病灶形成能力。结果 克隆形成法可以有效在子宫内膜异位症患者在位子宫内膜功能层中收集具有强克隆形成能力的克隆形成细胞。相较于非克隆形成细胞，MTT法、连续克隆形成实验、微球体形成实验显示克隆形成细胞具有更强增殖能力，连续克隆形成实验显示克隆形成细胞具有更强克隆形成能力，微球体形成实验显示克隆形成细胞具有更强的增殖及自我更新能力，诱导分化实验显示克隆形成细胞具有定向分化能力，裸鼠异位病灶形成实验显示克隆形成细胞具有更强的异位病灶形成能力。结论 子宫内膜异位症在位子宫内膜功能层中具有强克隆形成能力的子宫内膜干细胞，应用克隆形成法可以有效收集子宫内膜干细胞，经该法收集的子宫内膜干细胞可以冻存及传代培养，且具有干细胞无限增殖、自我更新、定向分化及异位病灶形成能力。

关键词: 子宫内膜异位症, 子宫内膜干细胞, 在位子宫内膜, 克隆形成

Abstract: Objective: to get the endometrium stem cells (ESC) for the eutopic endometrial from women with endometriosis (EMs) by using Colony-forming assay and provide the experimental basis for the further study of EMs. Study design: The endometrial samples from women with EMs undergoing fractional curettage respectively. Colony-forming cells (CFCs) were isolated from individual large colony-forming units (CFU) which visible macroscopically and contained >4000 cells. We focused on the difference of CFCs characteristics in women with EMs, including high proliferative potential（MTT assay）, serial clone formation, mammosphere formation of self-renewal, differentiation potential in vitro, and tissue reconstitution in vivo. Result: the optical densities in cell proliferation were more significant higher in the CFCs than N-CFCs ), The CE of CFCs from P1-5 are higher than N-CFCs 。There are statistical significances between the two groups (P<0.05). CFCs originated spheres with a diameter were at least 60μm, spheres were continuously generated at 5-6 day intervals for at least 3 passages with a bigger and bigger size，in contrast, the spheres of N-CFCs did not change much in size after plating for 1 passage, and cannot be passaged serial .The protein of CFCs was negative for CK-19 before induction culture, but were expressed at CFCs, which cultured with the induction medium for 21 days. The endometriotic lesion-forming efficiency (100%) in the CFCs group was higher than that of N-CFCs group(40.63%), when CFCs and N-CFCs were inoculated into the abdominal subcutaneous of nude mice, respectively. Reconstruction of endometriotic lesions in the CFCs group with an average volume at (30.95 ±12.13)mm3 was bigger than the N-CFCs group (19.12±10.98)mm3. Conclusion: There are stem cells (SC) in the function layer of endometrium in EMs patients and it can be isolated from eutopic endometrium using colony-forming assays. The study has given a simple method to get ESC easily for researching EMs.

Key words: Endometriosis, Endometrial stem cells, Eutopic endometrial, Colony-forming assay