解剖学报

解剖学报 ›› 2019, Vol. 50 ›› Issue (4): 438-444.doi: 10.16098/j.issn.0529-1356.2019.04.006

• 细胞和分子生物学 • 上一篇    下一篇

 从异位子宫内膜分离获得的内膜干细胞的生物学特点

梁盛英1 王红梅2 杨芬1,4 孙钰椋3 刘彦礼1,3 杨君2* 林俊堂 1,3,4*    

  1. 1.河南省医用组织再生重点实验室; 2.新乡医学院第一附属医院妇二科; 3.新乡医学院生命科学技术学院干细胞中心;4.新乡医学院生物医学工程学院, 河南 新乡 453003
  • 收稿日期:2018-08-01 修回日期:2018-11-14 出版日期:2019-08-06 发布日期:2019-08-06
  • 通讯作者: 杨君;林俊堂 E-mail:linjtlin@126.com
  • 基金资助:
    国家自然科学基金课题;新乡市科技重大专项;河南省神经损伤修复重点实验室开放课题;新乡医学院产学研项目;新乡医学院研究生科研创新支持计划资助项目

Biological characteristics of endometrial stem cells isolated from ectopic lesions of endometriosis

 LIANG Sheng-ying1 WANG Hong-mei2 YANG Fen 1,4 SUN Yu-liang3 LIU Yan-li 1,3 YANG Jun 2* LIN Jun-tang 1,3,4*   

  1. 1.He’nan Key Laboratory of Medical Tissue Regeneration; 2.The First Affiliated Hospital of Xinxiang Medical University;  3.Stem Cell Research Center, College of Life Science and Technology, Xinxiang Medical University; 4. College of Biomedical Engineering, Xinxiang Medical University, He’nan Xinxiang 453003, China
  • Received:2018-08-01 Revised:2018-11-14 Online:2019-08-06 Published:2019-08-06
  • Contact: YANG Jun;LIN Jun-tang E-mail:linjtlin@126.com

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摘要：

目的 建立子宫内膜异位症（内异症）患者异位病灶来源子宫内膜干细胞（EnSCs）的分离培养及鉴定方法，并对所分离细胞的生物学特性进行检测，为研究EnSCs在内异症发病机制中的潜在作用提供支持。 方法 收集临床内异症患者异位病灶内膜组织样本，机械剪碎后Ⅰ型胶原酶消化，分离获得异位EnSCs，常规培养传代（n=10）;免疫荧光检测细胞波形蛋白（vimentin）的表达（n=3）; MTT法检测细胞增殖活性（n=5）;成脂成骨诱导检测细胞多向分化潜能（n=3），流式细胞仪检测细胞表面标志物（n=3）;蛋白芯片检测细胞条件培养基中促血管再生因子及相关炎症因子的分泌（n=6），同时检测细胞表面相关黏附分子的表达（n=7）。 结果 成功从异位病灶中分离获得异位EnSCs，所分离细胞强阳性表达骨架蛋白vimentin，且细胞阳性表达间充质干细胞表面标志物CD29、CD73、CD90及CD105，阴性表达造血干细胞表面标志物CD34和CD45；经成脂诱导分化后油红O染色可见明显脂滴，成骨诱导分化后茜素红染色可见明显钙结节；蛋白芯片检测结果表明，异位EnSCs能够分泌高浓度的促血管再生因子血管内皮生长因子(VEGF)、血管紧张素(ANG)及血小板衍生生长因子 (PDGF)-AA，以及与促血管再生密切相关的炎症因子白细胞介素 (IL)-6、IL-8和单核细胞趋化蛋白-1(MCP-1)，且细胞表面高表达活化白细胞黏附分子（ACLAM)和细胞间黏附分子-1（ICAM-1）。 结论 本研究成功建立内异症患者异位病灶来源EnSCs的分离培养及鉴定方法；对其活性因子分泌的研究表明，异位EnSCs具有明显的促血管再生作用。上述实验结果肯定了内异症患者异位病灶中干细胞的存在，在支持内异症发病机制中“干细胞学说”的同时，为进一步围绕异位EnSCs开发潜在的内异症治疗靶点提供可能。

关键词: 子宫内膜异位症, 旁分泌作用, 黏附因子, 免疫荧光, 流式细胞术, 异位子宫内膜干细胞

Abstract:

Objective To establish the approach of isolating, culturing and identifing endometrial stem cells (EnSCs) derived from ectopic lesion of endometriosis patient; and preliminarily examine the biological characteristic of ectopic EnSCs, which provide support for further study on the potential role of ectopic EnSCs in the pathogenesis of endometriosis. Methods The ectopic lesions of endometriosis were harvested from the patients with the informed consent and transferred to lab as soon as possible. The ectopic lesions were minced, digested by collagenase Ⅰ and seeded into cell culture flasks for conventional culture (n=10). Expression of vimentin in ectopic EnSCs was examined by immunofluorescence (n=3). Proliferative capacity of ectopic EnSCs was examined by MTT assay (n=5). Multilineage differentiation potential of ectopic EnSCs was examined by adipogenic and osteogenic differentiation respectively (n=3). Immunophenotype analysis of ectopic EnSCs was determined by flow cytometry (n=3). Production of biological factors in ectopic EnSCs derived conditional medium (n=6) and expression of adhesion molecules on ectopic EnSCs (n=7) were examined by protein assays. Results We successfully isolated EnSCs from ectopic lesions of endometriosis patients, and ectopic EnSCs were positive for vimentin and typical markers of mesenchymal stem cell (CD29, CD73, CD90 and CD105), and negative for the markers of hematopoietic stem cell (CD34 and CD45). The induced ectopic EnSCs showed obvious lipid droplets (adipogenic differentiation) and calcium nodules (osteogenic differentiation). The ectopic EnSCs could secrete high concentration of angiogenic factors ［vascular endothelial growth factor (VEGF), angiotensin (ANG) and platelet-derived growth factor (PDGF)-AA］and angiogenesis associated inflammation cytokines ［interleukin (IL-6)，IL-8 and monocyte chemotactic protein 1(MCP-1)］. Additionally, adhesion molecules analysis demonstrated the high expression of activated leukocyte adhesion molecule（ACLAM）and intercellular cell adhesion molecule-1（ICAM-1）on ectopic EnSCs. Conclusion We successfully establish the procedure of isolating and culturing ectopic EnSCs and demonstrate that ectopic EnSCs is capable of promoting angiogenesis through secreting high concentration of associated biological factors. The above result confirm the existence of EnSCs in ectopic lesions of endometriosis, which not only supports the stem cell based pathogenesis of endometriosis, but also shows the therapeutic potential of taking ectopic EnSCs as promising targets in the treatment of endometriosis.

Key words: Endometriosis, Paracrine effect, Adhesion factor, Immunofluorescence, Flow cytometry, Ectopic endometrial stem cell