经血源性子宫内膜间充质干细胞的分离、培养与鉴定  

Isolation,culture and identification of menstrual blood-derived mesenchymal stem cells

作　　者：周云帆[1,2] 杨波[2,3] 胡祥[4,5] 焦洪亮[2,3] 周长辉[1] 田毅[2,3] 雷宁静[1] 谷晨熙[6] 许予明[7] 戴建武[8] 关方霞[1] 

机构地区：[1]郑州大学生物工程系,河南省郑州市450001 [2]河南省高等学校临床医学重点学科开放实验室,河南省郑州市450052 [3]郑州大学第一附属医院神经外科,河南省郑州市450052 [4]江苏省干细胞与生物治疗公共技术服务平台,江苏省泰州市225300 [5]深圳市北科细胞工程研究所,广东省深圳市518000 [6]郑州大学临床医学系,河南省郑州市450001 [7]郑州大学第一附属医院神经内科,河南省郑州市450052 [8]中国科学院遗传与发育生物学研究所,北京市100190

出　　处：《中国组织工程研究与临床康复》2010年第32期5952-5956,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：郑州大学211三期建设项目"干细胞基础与临床研究;河南省科技厅2009科技发展计划项目(092102310250);河南省青年骨干教师资助项目;郑州市二七区科技攻关计划项目(20095157);江苏省干细胞与生物治疗公共技术服务平台项目(BM2008146)~~

摘　　要：背景:目前用于干细胞研究的主要来源为胚胎干细胞和骨髓干细胞,但这两种来源都存在着部分局限性,因此需要寻找一种新型的干细胞以克服肿瘤形成的潜在性、标本来源的缺乏及伦理学的争议等弊端。目的:分离培养经血源性子宫内膜间充质干细胞并进行鉴定。方法:采用Ficoll密度梯度离心法分离出人经血源性子宫内膜间充质干细胞,对细胞的形态及生长特性进行观察;采用流式细胞仪对细胞表面抗原CD29、CD44、CD34、CD45、HLA-ABC及HLA-DR进行表型鉴定;免疫细胞化学法测定经血源性子宫内膜间充质干细胞中nestin阳性细胞表达。结果与结论:应用Ficoll密度梯度离心法可以从女性月经血中分离培养出干细胞,约2周原代细胞达到80%～90%融合,细胞呈漩涡状、网状、辐射状。传代后能稳定生长,可见纤维样细胞形态为主导。流式检测结果显示,经血源性子宫内膜间充质干细胞表现出CD29强阳性,CD44阳性,CD34、CD45、HLA-DR阴性,低表达HLA-ABC。染色显示nestin抗原在经血源性子宫内膜间充质干细胞中有表达,约占经血源性子宫内膜间充质干细胞的(10.35±0.51)%。结果表明经血源性子宫内膜间充质干细胞表达间充质干细胞的标记物,具低免疫源性;经血源性子宫内膜间充质干细胞表达nestin抗原,为经血源性子宫内膜间充质干细胞在神经系统应用提供一定的理论依据。BACKGROUND：Generally,the main sources of stem cells are embryonic stem cells and bone marrow stem cells,but there are some limitations of the two kinds of sources,and researchers strive to find a new type of stem cells in order to overcome the potential for teratoma,the lack of sample sources,ethics controversies and so on. OBJECTIVE：To isolate,culture and identify menstrual blood-derived mesenchymal stem cells （MMCs）. METHODS：MMCs were separated by Ficoll density gradient centrifugation. Cell morphology and growth characteristics were observed. Flow cytometry analysis was used to identify expression of CD29,CD44,CD34,CD45,HLA-ABC and HLA-DR surface markers in MMCs. Immunocytochemical method was utilized to detect nestin-positive cells in MMCs. RESULTS AND CONCLUSION：MMCs were successfully separated from menstrual blood by Ficoll density gradient centrifugation. About 2 weeks later,primary cells reached 80% to 90% confluence; cells were spiral-shaped,mesh and radial. After passage to a stable growth,cells could be seen as the leading fiber-like cell morphology. Results from flow cytometry showed that MMCs were highly positive for CD29,positive for CD44,low positive for HLA-ABC,but negative for CD34,CD45 and HLA-DR. Staining showed nestin expression of antigen in MMCs was about （10.35±0.51）%. Results have suggested that MMCs express MSCs markers,and only have low immunogenicity. MMCs express nestin antigen and provide some theoretical basis for MMCs in the nervous system application.

关 键 词：经血 子宫内膜 间充质干细胞 表型鉴定 巢蛋白 

分 类 号：R394.2[医药卫生—医学遗传学]