北京生物医学工程

猪骨髓间质干细胞分离培养及表面抗原鉴定

Isolation, Expansion, and Antigen Expression of Porcine Bone Marrow Mesenchymal Stem Cells

作者：韩志军任华

单位：中国医学科学院北京协和医学院 (北京100730)

分类号：

出版年·卷·期（页码）：2011·30·3（291-295）

摘要：

目的建立猪骨髓间质干细胞(bone marrow mesenchymal stem cells, BMMSC)体外分离培养、纯化的方法，观察其生物学特性，为其作为组织工程学种子细胞提供实验基础。方法取猪的胸骨骨髓6～8ml,用Ficoll淋巴细胞分离液提取单核细胞,采用贴壁法培养。接种后2h利用倒置显微镜观察细胞的大体形态，并利用流式细胞仪检测细胞抗原表达。结果原代培养接种3～4h后细胞开始贴壁，24h后贴壁细胞数量明显增多，3~4d后细胞呈纺锤形且形成为单个或数个细胞克隆，7～9d后集落迅速增多，细胞融合，在10～12d细胞铺满全层。流式细胞仪监测到BMMSC表达CD29、CD44、CD105、KDR，不表达HLA-DR、CD11a、CD14、CD31、CD34、CD45。结论采用密度梯度离心法培养猪的BMMSC生长稳定,增殖能力强，具有间质干细胞的表面抗原特征，该方法可作为培养猪BMMSC的常规方法。

Objective To establish the methods of isolation, expansion, and purification of porcine bone marrow mesenchymal stem cells (BMMSC) in vitro, and provide the experimental basis for BMMSC as tissue engineering seed cells by observing its biology characteristic. Method A volume of 6-8ml porcine bone marrow was aspirated from sternum. The mononuclear cells were harvested using Ficoll lymphocyte separating medium at a density of 1.077. the BMSC were cultured by the adherence method. Cellular morphology was observed by microscope, and cellular surface antigen expression was examined by the instrument flow cytometry. Result After seeded 3-4 hours, cells adhered to the bottom. Twenty-four hours later, the number of the adhered cells significantly increased, and 3-5 days later, the primary cultured cells were in uniformly long spindle-shaped form and formed cell-colony. After seeded 7-9 days, the cell-colonies rapidly increased, and 10-12 days later, cells spread to the bottom. The test of Flow cytometry showed that the BMMSC were positive for CD29,CD44,CD105 and KDR, while negative for HLA-DR, CD11a, CD14, CD31, CD34 or CD45. Conclusion By density gradient centrifugation and adherence method, BMMSC of porcine were obtained easily and proliferate rapidly, which showed the antigen characteristics of mesenchymal stem cell. The proposal method could be used as a routine method for BMMSC preparation.

参考文献：

［1］Delorme B, Ringe J, Pontikoglou C, et al. Specific lineage-priming of bone marrow mesenchymal stem cells provides the molecular framework for their plasticity［J］. Stem Cells， 2009,27(5):1142-1151.
［2］Yoon J, Min BG, Kim YH, et al.Differentiation, engraftment and functional effects of pre-treated mesenchymal stem cells in a rat myocardial infarct model［J］. Acta Cardiol, 2005,60(3):277-284.
［3］Friendenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiate mouse hematopoietic organs［J］. Exp Hematol 1976,4(5): 267-274.
［4］Gnecchi M, Melo LG. Bone marrow-derived mesenchymal stem cells: isolation, expansion, characterization, viral transduction, and production of conditioned medium［J］. Methods Mol Biol, 2009,482:281-294.
［5］Roubelakis MG, Pappa KI, Bitsika V. Molecular and proteomic characterization of human mesenchymal stem cells derived from amniotic fluid: comparison to bone marrow mesenchymal stem cells［J］. Stem Cells Dev, 2007,16(6):931-952.
［6］Perdikogianni C, Dimitriou H, Stiakaki E. Could cord blood be a source of mesenchymal stromal cells for clinical use? Cytotherapy, 2008,10(5):452-459.
［7］Tocci A, Laura FI. Mesenchymal stem cell: use and perspectives［J］. J Hematol,2003,4: 92-96.
［8］Park PC, Selvarajah S, Bayani J. Stem cell enrichment approaches［J］. Semin Cancer Biol, 2007,17(3):257-264.
［9］Horn P, Bork S, Diehlmann A. Isolation of human mesenchymal stromal cells is more efficient by red blood cell lysis［J］. Cytotherapy, 2008,10(7):676-685.
［10］Mosna F, Sensebé L, Krampera M. Human Bone-Marrow And Adipose Tissue Mesenchymal Stem Cells: A User’s Guide［J］. Stem Cells Dev, 2010,19(10):1449-1470.
［11］Beyer Nardi N, da Silva Meirelles L. Mesenchymal stem cells: isolation, in vitro expansion and characterization［J］. Handb Exp Pharmacol,2006,174: 249-282.
［12］Pontikoglou C, Delorme B, Charbord P. Human bone marrow native mesenchymal stem cells［J］. Regen Med,2008,3(5):731-741.

服务与反馈：

【文章下载】【加入收藏】

提示：您还未登录，请登录！点此登录