中国组织工程研究 ›› 2021, Vol. 25 ›› Issue (在线): 4970-4975.doi: 10.12307.2021.000

• • 上一篇    

人胎盘源间充质干细胞条件培养液可上调缺氧状态下BeWo细胞活力和紧密连接因子的表达

林清凡1,解一新2,陈婉清1,叶振忠3,陈幼芳1   

  1. 1泉州医学高等专科学校,福建省泉州市  362011;2石狮市(华侨)医院,福建省石狮市  362700;3福建领航干细胞科技有限公司,福建省泉州市  362011
  • 收稿日期:2020-06-23 修回日期:2020-07-01 接受日期:2020-08-25 出版日期:2021-01-28 发布日期:2021-01-20
  • 通讯作者: 陈幼芳,博士,教授,泉州医学高等专科学校,福建省泉州市 362011
  • 作者简介:林清凡,男,1987年生,福建省厦门市人,2015年福建师范大学毕业,硕士,研究实习员,主要从事肿瘤细胞、干细胞相关的分子生物学与基因组学研究。
  • 基金资助:
    福建省母婴健康服务应用技术协同创新中心基金(XJM1804),项目负责人:林清凡;福建省自然科学基金(2016J01422),项目负责人:陈幼芳;泉州市科技计划项目(2016Z005),项目参与人:林清凡

Human placenta-derived mesenchymal stem cell conditioned medium can upregulate BeWo cell viability and zonula occludens expression under hypoxia

Lin Qingfan1, Xie Yixin2, Chen Wanqing1, Ye Zhenzhong3, Chen Youfang1   

  1. 1Quanzhou Medical College, Quanzhou 362011, Fujian Province, China; 2Shishi General Hospital, Shishi 362700, Fujian Province, China; 3Fujian Genesis Stem Cell Co., Ltd., Quanzhou 362011, Fujian Province, China 
  • Received:2020-06-23 Revised:2020-07-01 Accepted:2020-08-25 Online:2021-01-28 Published:2021-01-20
  • Contact: Chen Youfang, MD, Professor, Quanzhou Medical College, Quanzhou 362011, Fujian Province, China
  • About author:Lin Qingfan, Master, Quanzhou Medical College, Quanzhou 362011, Fujian Province, China
  • Supported by:
    the Maternal and Child Health Service Application Technology Collaborative Innovation Center Fund of Fujian Province, No. XJM1804 (to LQF); the Natural Science Foundation of Fujian Province, No. 2016J01422 (to CYF); the Science and Technology Project of Quanzhou, No. 2016Z005 (to LQF) 

摘要:

文题释义:

间充质干细胞条件培养液:间充质干细胞生长过程中会分泌生长因子、细胞因子等多种活性物质到培养液中。通过收集该类培养液,经过滤或浓缩等处理,获得不含细胞成分但含有该间充质干细胞分泌物的培养液,就称为间充质干细胞条件培养液。
紧密连接:是细胞间连接方式之一,其由跨膜蛋白紧密连接黏附分子、咬合蛋白、闭合蛋白和胞质分子闭合小环蛋白1等构成紧密连接蛋白复合物,从而形成分支状封闭索将两相邻细胞膜紧贴,封闭细胞间空隙,形成细胞间紧密相连区域。它除有机械连接作用外,更是组织屏障构成的关键,如胎盘屏障中滋养层与血管内皮细胞间存有大量紧密连接结构。

背景:近年来间充质干细胞已是新药开发领域的研究热点,其旁分泌效应也已应用于多种组织损伤性细胞模型修复评估实验,但目前尚无研究探索其条件培养液是否对缺氧状态下人胎盘滋养层系细胞的紧密连接功能具有修复作用。
目的:探讨人胎盘源间充质干细胞条件培养液对缺氧条件下人胎盘绒毛膜癌滋养层系BeWo细胞活力和紧密连接因子表达的影响。
方法:采用1 000 μmol/L氯化钴处理12 h诱导BeWo细胞以构建胎盘屏障缺氧模型,检测细胞活力以及缺氧诱导因子1α和紧密连接因子闭合小环蛋白1、封闭蛋白4、封闭蛋白8的表达量变化;在缺氧模型中添加人胎盘源间充质干细胞条件培养液建立缺氧干预组、复氧修复组,检测细胞活力和上述4个基因的表达量改变;为探讨人胎盘源间充质干细胞条件培养液改变闭合小环蛋白1表达量的潜在机制,建立胰岛素样生长因子1缺氧干预组、胰岛素样生长因子1处理组,检测BeWo细胞活力以及闭合小环蛋白1表达量。
结果与结论:①氯化钴诱导缺氧显著下调BeWo细胞活力,上调缺氧诱导因子1α mRNA和蛋白表达,下调闭合小环蛋白1和封闭蛋白4 mRNA和蛋白表达,上调封闭蛋白8 mRNA表达,但其蛋白表达下降;②人胎盘源间充质干细胞条件培养液明显提升缺氧条件下BeWo细胞活力和闭合小环蛋白1的表达,下调缺氧诱导因子1α的表达,但对封闭蛋白4无作用;③胰岛素样生长因子1干预缓解了缺氧对BeWo细胞活力的抑制作用,并上调闭合小环蛋白1的表达。
https://orcid.org/0000-0002-5183-0245(林清凡) 

关键词: 干细胞, 人胎盘源间充质干细胞, 条件培养液, 氯化钴, 缺氧, BeWo细胞, 闭合小环蛋白1

Abstract: BACKGROUND: In recent years, mesenchymal stem cells have become a research hotspot in the field of new drug development, and their paracrine effect has also been applied in the repair evaluation experiments of a variety of tissue injured cell models, but there is no research to explore whether their conditioned medium can repair the tight junction function of human placental trophoblast cells under hypoxia.
OBJECTIVE: To demonstrate the effect of mesenchymal stem cell conditioned medium on cell viability and expression of tight junction factors in human placental choriocarcinoma trophoblast cell line BeWo under hypoxia. 
METHODS: BeWo cells were exposed to hypoxia induced by 1 000 μmol/L CoCl2 for 12 hours to construct the hypoxia model of placental barrier. For model evaluation, five indexes were measured, including cell viability and expression of hypoxia induced factors-1α, as well as three tight junction factors: zonula occludens 1, claudins 4 and claudins 8. Mesenchymal stem cell conditioned medium was added into hypoxia model to construct hypoxia intervention and reoxygenation groups. To investigate the potential mechanism of zonula occludens 1 expression recovery induced by mesenchymal stem cell conditioned medium, insulin-like growth factor 1 hypoxia intervention and treatment groups were established, and the BeWo cell viability and zonula occludens 1 expression were detected.   
RESULTS AND CONCLUSION: (1) BeWo cells viability, zonula occludens 1 and claudins 4 expression were reduced by CoCl2-mimic hypoxia, with hypoxia induced factors-1α mRNA and protein upregulation. However, the expression of claudins 8 mRNA was upregulated, and its protein was downregulated. (2) Human placenta-derived mesenchymal stem cell conditioned medium improved BeWo cells viability and recovered expression level of hypoxia induced factors-1α and zonula occludens 1 under hypoxia induced by CoCl2, but it has no effect on claudins 4. (3) Insulin like growth factor-1 intervention alleviated the inhibition of hypoxia on the viability of BeWo cells, and upregulated the expression of zonula occludens 1. 

Key words: stem cells, human placenta-derived mesenchymal stem cells, conditioned medium, CoCl2, hypoxia, BeWo cells, zonula occludens 1

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