中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (33): 5293-5299.doi: 10.3969/j.issn.2095-4344.1830

• 胚胎干细胞 embryonic stem cells • 上一篇    下一篇

人胎盘间充质干细胞的扩增与鉴定

马  晨1,2,李晓国2,徐明均2,马晓娜2,马晓薇3   

  1. 1宁夏医科大学临床医学院,宁夏回族自治区银川市  750003;2宁夏人类干细胞研究所,宁夏回族自治区银川市  750004;3宁夏医科大学总医院ICU,宁夏回族自治区银川市  750012
  • 修回日期:2019-06-06 出版日期:2019-11-28 发布日期:2019-11-28
  • 通讯作者: 马晓薇,博士,主任医师,教授,硕士生生导师,宁夏医科大学总医院ICU,宁夏回族自治区银川市 750012
  • 作者简介:马晨,男,1989年生,宁夏回族自治区石嘴山市人,回族,宁夏医科大学在读硕士研究生,主要从事急性呼吸衰竭方面的研究。
  • 基金资助:

    国家自然科学基金(81860350),项目负责人:马晓薇;宁夏自然科学基金(2018A0705),项目负责人:马晓薇;宁夏高等院校科学研究项目(NGY2017126),项目负责人:马晓薇

Amplification and identification of human placental mesenchymal stem cells

Ma Chen1, 2, Li Xiaoguo2, Xu Mingjun2, Ma Xiaona2, Ma Xiaowei3   

  1. 1School of Clinical Medicine, Ningxia Medical University, Yinchuan 750003, Ningxia Hui Autonomous Region, China; 2Ningxia Human Stem Cell Institute, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 3Intensive Care Unit, General Hospital of Ningxia Medical University, Yinchuan 750012, Ningxia Hui Autonomous Region, China
  • Revised:2019-06-06 Online:2019-11-28 Published:2019-11-28
  • Contact: Ma Xiaowei, MD, Chief physician, Professor, Master’s supervisor, Intensive Care Unit, General Hospital of Ningxia Medical University, Yinchuan 750012, Ningxia Hui Autonomous Region, China
  • About author:Ma Chen, Master candidate, School of Clinical Medicine, Ningxia Medical University, Yinchuan 750003, Ningxia Hui Autonomous Region, China; Ningxia Human Stem Cell Institute, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • Supported by:

    the National Natural Science Foundation of China, No. 81860350 (to MXW); the Natural Science Foundation of Ningxia Hui Autonomous Region, No. 2018A0705 (to MXW); Scientific Research Project of Ningxia Higher Education, No. NGY2017126 (to MXW)

摘要:

文章快速阅读:

文题释义:
人胎盘间充质干细胞(hpMSC):
来源于人胎盘胎儿侧中胚层,和其他间充质干细胞一样具有低免疫原性、多向分化、损伤趋化作用,同时其容易获得,为医学科研工作提供了数量可观的间充质干细胞,人胎盘间充质干细胞已运用于多种疾病的科研中并取得了不错的效果。
人胎盘间充质干细胞三系体外诱导分化:体外培养人胎盘间充质干细胞,待细胞稳定后更换细胞培养基,向中胚层细胞诱导分化,即向骨、软骨、脂肪细胞诱导分化,经过21 d的体外诱导分化,分别用茜素红、甲苯胺蓝、油红O进行细胞染色观察人胎盘间充质干细胞向中胚层细胞的分化能力。

 

摘要
背景:
作为组织工程的种子细胞,胎盘间充质干细胞因具有多向分化、低免疫原性、免疫调节等作用,被广泛用于临床疾病的治疗及临床科研中。
目的:根据间充质干细胞的定义来鉴定从宁夏医科大学总医院干细胞研究所获得的人胎盘间充质干细胞,为后续的实验提供可靠的保障。
方法:培养从宁夏医科大学总医院干细胞研究所获得的人胎盘间充质干细胞,观察其在塑料培养皿或培养瓶内的贴壁情况,并间断观察细胞形态发生的变化。将培养的第3代人胎盘间充质干细胞通过流式细胞技术检测细胞表面特异性抗原,并且用成脂、成骨、成软骨诱导培养基分别诱导21 d后,采用油红O、茜素红、甲苯胺蓝进行细胞染色观察人胎盘间充质干细胞向脂肪、骨,软骨细胞的分化能力。实验方案得到宁夏医科大学总医院医学科研伦理审查委员会批准(编号:2018-047)。
结果与结论:①经过体外培养的第3代人胎盘间充质干细胞呈漩涡状贴壁生长,单个细胞呈梭状,形似纤维细胞;②人胎盘间充质干细胞具有特殊的细胞免疫学表型:流式细胞术检测CD105、CD90、CD73呈强阳性表达;造血标记物CD34、CD45、CD14以及HLA-DR呈阴性表达;③人胎盘间充质干细胞在成脂、成骨、成软骨诱导培养21 d后,油红O染色、茜素红染色及甲苯胺蓝染色均呈阳性。提示:根据间充质干细胞的定义,来自宁夏医科大学总医院干细胞研究所的人胎盘间充质干细胞符合间充质干细胞的特性,为后续实验提供良好保证。


中国组织工程研究杂志出版内容重点:干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程
ORCID: 0000-0003-4313-5768(马晨)

关键词: 人胎盘间充质干细胞, hpMSC, 传代培养, 成脂诱导, 成骨诱导, 成软骨诱导, 流式细胞术

Abstract:

BACKGROUND: As a seed cell for tissue engineering, placental mesenchymal stem cells are widely used in the treatment of clinical diseases and clinical research due to their multi-directional differentiation, low immunogenicity, and immunomodulation.
OBJECTIVE: To identify human placental mesenchymal stem cells from the Stem Cell Research Institute of Ningxia Medical University General Hospital according to the definition of mesenchymal stem cells, and to provide reliable protection for subsequent experiments.
METHODS: The human placental mesenchymal stem cells were obtained from the Stem Cell Research Institute of Ningxia Medical University General Hospital. Cells attached to plastic culture dishes or culture flasks were detected, and cell morphology was observed intermittently. The cultured third-generation human placental mesenchymal stem cells were tested for cell surface specific antigen by flow cytometry, and were cultured in adipogenic, osteogenic and chondrogenic induction media, respectively. After 21 days of induction, Oil red O, alizarin red and toluidine blue were used for cell staining to observe the differentiation ability of human placental mesenchymal stem cells into fat, bone and chondrocytes. The study protocol was approved by the Ethics Committee of Ningxia Medical University General Hospital (approval No. 2018-047).
RESULTS AND CONCLUSION: The third-generation human placental mesenchymal stem cells cultured in vitro showed a vortex-like adherent growth. The single cell was fusiform with fibroblast-like appearance. Human placental mesenchymal stem cells had a special cellular immunological phenotype: CD105, CD90, and CD73 were positively expressed, and CD34, CD45, CD14 and HLA-DR were negatively expressed. The human placental mesenchymal stem cells were induced to differentiate in vitro after 21 days, and were positive for alizarin red, oil red O, and toluidine blue staining. As per the definition of mesenchymal stem cells, human placental mesenchymal stem cells from the Stem Cell Research Institute of Ningxia Medical University General Hospital meet the characteristics of mesenchymal stem cells, providing good guarantee for further experiments.

Key words: human placental mesenchymal stem cells, hpMSC, subculture, adipogenic induction, osteogenic induction, chondrogenic induction, flow cytometry

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