差速贴壁法分离培养早期人胚骨髓间充质干细胞-东南大学学报医学版

差速贴壁法分离培养早期人胚骨髓间充质干细胞

单位：1.东南大学附属中大医院,神经外科,江苏,南京,210029； 2.苏州大学医学院,神经生物学教研室,江苏,苏州,215005； 3.苏州大学附属第一医院,神经外科,江苏,苏州,215006

关键词：骨髓基质细胞/细胞学间充质干细胞/细胞学间充质干细胞/胚胎学细胞培养/方法细胞分化人类

分类号：R-332, R329.2

出版年·卷·期（页码）：2005·24·第四期（218-221）

摘要：

目的:探讨差速贴壁法从早期人胚股骨分离、纯化骨髓间充质干细胞(MSCs)的可行性,并观察培养细胞是否向神经元样细胞诱导分化.方法:取2～3月龄新鲜健康流产人胚股骨,切碎,接种玻璃培养瓶内培养8～12 h,待较多的成纤维细胞贴壁,立刻将未贴壁细胞转种至塑料培养瓶培养,并多次传代;用甲氧酚(BHA)和二甲亚砜(DMSO)诱导培养的细胞,免疫细胞化学方法检测诱导后细胞神经元烯醇化酶(NSE)、神经巢蛋白(Nestin)、胶质纤维酸性蛋白(GFAP)表达情况.结果:原代培养3 d可见塑料培养瓶内有长梭形细胞生长,至第10天前后形成集落样克隆,传至第4代后呈单层漩涡状排列的长梭形的成纤维样细胞,已无明显细胞克隆形成,经多次传代,细胞保持良好的增殖能力和稳定的性状;免疫细胞化学方法显示未诱导细胞NSE、Nestin、GFAP阴性,诱导后细胞NSE、Nestin呈阳性,GFAP阴性,间接证实培养所得细胞是MSCs.结论:差速贴壁培养法从早期人胚股骨中分离纯化MSCs,去除可能的成纤维细胞污染,是简便有效的,可供有关研究参考选择.

Objective To investigate the feasibility of expansion and purification of bone marrow mesenchymal stem cells (MSCs) derived from human fetal thighbone in vitro and their differentiation into neuron cells. Methods Cells were isolated by differential adhesion from human fetal thighbone and expanded in culture medium. MSCs were induced to differentiate into neuron-like cells with dimethylsulfoxide (DMSO) and butylated hydroxyanisole (BHA). Specific markers were detected by immunohistochemistry. Results MSCs were purified and expanded to be undifferentiated cells in vitro with the culturing method. Immunohistochemistry staining showed that the induced MSCs expressed neuronspecific enolase (NSE) and nestin, but did not express glial fibrillary acidic protein (GFAP). Conclusion This culturing method of differential attachment is a reliable method of primary culturing the isolated MSCs from human fetal thighbone successfully, which can be used for further basic research and clinical applications.

参考文献：

[1] Ohgushi H, CAPLAN A I. Stem cell technology and bioceramics:from cell to gene engineering, 1999
[2] 钱新华, 朱为国, 赵彤. 异基因造血干细胞移植并发致死性间质性肺炎, 1997(12)
[3] THOMOSON J A, ITSKOVITZ-ELDOR J, SHAPIRO S S. Embryonic stem cell lines derived from human blastocysts. 1998. doi:10.1126/science.282.5391.1145
[4] KREIDER B Q, MESSING A, DOAN H. Enrichment of schwann cell cultures from neonatal rat sciatic nerve by differential adhesion. 1981(2). doi:10.1016/0006-8993(81)90375-9
[5] Sanchez- Ramos J, SONG S, CARDOZO- PELAEZ F. Adult bone marrow stromal cells differentiate into neural cells in vitro. 2000
[6] Biancoa P, RIMINUCCIB M, GRONTHOSC S. Bone marrow stromal stem cells:nature,biology,and potential applications. 2001(3). doi:10.1634/stemcells.19-3-180
[7] 杨立业, 郑佳坤, 刘相名. 脂肪组织来源的基质细胞向神经元样细胞分化. 四川大学学报(医学版)2003(3). doi:10.3969/j.issn.1672-173X.2003.03.002
[8] 陈镭, 惠国桢, 栾文忠. 脐带血间充质干细胞体外分离、纯化及培养. 实用临床医学2004(8). doi:10.3969/j.issn.1672-2353.2004.01.004
[9] 金钧, 单立冬, 惠国桢. 人骨髓间充质干细胞的分离培养和向神经细胞分化的研究. 实用临床医药杂志2003(7). doi:10.3969/j.issn.1672-2353.2003.03.003
[10] Tse W T, PENDLETON J D, BEYER W M. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. 2003(3). doi:10.1097/01.TP.0000045055.63901.A9
[11] HITT M, BETT A J, ADDISON C L, at al. Techniques for human adenovirus vector construction and characterization: methods in molecular genetics, 1995
[12] Stute N, FEHSE B, SCHRODER J. Human mesenchymal stem cells are not of donor origin in patients with severe aplastic anemia who underwent sex-mismatched allogeneic bone marrow transplant. 2002

服务与反馈：

【文章下载】【发表评论】【查看评论】【加入收藏】

提示：您还未登录，请登录！点此登录