人胎盘间充质干细胞的扩增与鉴定  

Amplification and identification of human placental mesenchymal stem cells

作　　者：马晨 李晓国 徐明均 马晓娜 马晓薇[3] Ma Chen;Li Xiaoguo;Xu Mingjun;Ma Xiaona;Ma Xiaowei(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;Intensive Care Unit,General Hospital of Ningxia Medical University,Yinchuan 750012,Ningxia Hui Autonomous Region,China)

机构地区：[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

出　　处：《中国组织工程研究》2019年第33期5293-5299,共7页Chinese Journal of Tissue Engineering Research

基　　金：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~~

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。BACKGROUND: As a seed cell for tissue engineering, placental mesenchymal stem cells are widely used in the treatment of clinical diseasesand 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 UniversityGeneral 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 UniversityGeneral 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, andwere cultured in adipogenic, osteogenic and chondrogenic induction media, respectively. After 21 days of induction, Oil red O, alizarin red andtoluidine blue were used for cell staining to observe the differentiation ability of human placental mesenchymal stem cells into fat, bone andchondrocytes. 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-likeadherent growth. The single cell was fusiform with fibroblast-like appearance. Human placental mesenchymal stem cells had a special cellularimmunological phenotype: CD105, CD90, and CD73 were positively expressed, and CD34, CD45, CD14 and HLA-DR were negativelyexpressed. 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 theStem Cell Research Institute of Ningxia

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 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]