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《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:

21卷

期数:

2015年06期

页码:

525-529

栏目:

技术与方法

出版日期:

2015-11-20

文章信息/Info

Title:

?Comparison of adherence culture method and density gradient centrifugation method in isolating endometrical mensenchymal stem cells isolated from menstrual blood

文章编号:

1006-8147（2015）06-0525-05

作者:

袁 晴¹; 杜 雪¹; 袁碧波¹; 屈 野²; 周 园³; 石 慧³

（1．天津医科大学总医院妇产科，天津 300052；2.武警后勤学院病原生物与免疫学教研室，天津 300309；3.中国医学科学院北京协和医学院，血液学研究所血液病医院，实验血液学国家重点实验室，干细胞医学中心，天津 300020）

Author(s):

YUAN Qing ¹;  DU Xue¹;  YUAN Bi-bo;  ¹ QU Ye²;  ZHOU Yuan²;  SHI Hui³

（1．Department of Obstetrics and Gynecology，General Hospital , Tianjin Medical University , Tianjin 300052 , China；2 . Department of Pathogenic Biology and Immunology Logistics, College of Chinese People’s Armed Police Forces， Tianjin 300309,China;3. State Key Laboratory of Experimental Hematology, Institute of Hematology＆Blood Diseases Hospital, Chinese Academy of Medical Science ＆Peking Union Medical College, Tianjin 300020, China）

关键词:

间充质干细胞;  子宫内膜;  经血;  贴壁法;  密度梯度离心法

Keywords:

mesenchymal stem cell' target="_blank" rel="external">">mesenchymal stem cell;  endometrial;  menstrual blood; adherence culture; density gradient centrifugation method

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分类号:

Q813

DOI:

-

文献标志码:

A

摘要:

目的：比较贴壁法和密度梯度离心法对经血源性子宫内膜间充质干细胞（MB-MSCs）的分离效果。方法：选择25～35岁月经周期正常的健康女性的月经血，分别采用贴壁法和密度梯度离心法分离MB-MSCs并进行传代。镜下观察原代细胞形态变化；对比原代及第 2、3 代细胞传代时间；流式细胞仪检测表面标志物；阿尔辛兰染色检测其成软骨能力。 结果：贴壁法分离的原代细胞呈聚集样生长，密度梯度离心法分离的原代细胞6 d后也呈聚集样生长。贴壁法分离原代细胞的传代时间（14.58±1.31）d明显快于密度梯度离心法（19.17±1.34）d，差异有显著意义（P < 0.05）；第 2、3 代细胞的传代时间两种分离方法差异无统计学意义（P > 0.05）。经流式细胞仪检测，两种方法分离的MB-MSCs阳性表面标志物CD44 、CD29、 CD105、CD73含量和阴性表面标志物 CD31、CD45 含量比较差异无统计学意义（P > 0.05），两种方法分离的第 3 代细胞经成软骨诱导后比较无显著差别。结论：采用贴壁法与密度梯度离心法分离MB-MSCs效果相似。但贴壁法更适用于早期快速获得纯度较高的细胞。

Abstract:

Objective： To compare the effects of adherence culture method and density gradient centrifugation method in isolating m endometrical mensenchymal stem cells isolated from menstrual blood(MB-MSCs). Methods: MB-MSCs were obtained from healthy female volunteers aged from 25 to 35 years and isolated by adherence culture method and density gradient centrifugation method. Observation of Primary cell morphology characteristics, the generation time of the primary, 2nd and 3rd passage MB-MSCs were) compared between two methods and the surface markers were detected by flow cytometer. In addition, chondrogenic differentiation of MSCs was examined by Aldrich staining. Results： Primary cells isolated by adherence culture method showed aggregation growth, while cells isolated by density gradient centrifugation method showed diffusion growth. The generation time of primary cells isolated by adherence culture method (14.58±1.31) was significantly shorter than that of cells isolated by density gradient centrifugation method (19.17±1.34) days (P < 0.05), while the generation time of the 2nd and 3rd passage cells showed no statistically significant differences between these methods (P > 0.05). The content of positive surface markers CD44, CD29, CD105, CD73, CD44 and negative surface marker CD31, CD45 showed no significant difference between these two isolation methods (P > 0.05); no significant difference at Passage 3 was found by Aldrich staining in chondrogenic cells. Conclusion: MB-MSCs could be isolated by adherence culture method, and the cell isolation effects of adherence culture method are equal to that of density gradient centrifugation method. Adherence culture applies to early and rapid high purity cell culture methods. The method is simple, economical, and efficient than other methods and can reduce the chance of contamination to the lowest level.

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备注/Memo

备注/Memo:

基金项目 国家自然科学基金资助项目（81303108）

作者简介 袁晴（1989-）,女，硕士在读，研究方向：妇科肿瘤；通信作者 ：袁碧波 ，E-mail：yuanbibotj@163.com。

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更新日期/Last Update: 2015-11-27