Acta Academiae Medicinae Sinica

Acta Academiae Medicinae Sinica

Acta Academiae Medicinae Sinica ›› 2016, Vol. 38 ›› Issue (1): 9-15.doi: 10.3881/j.issn.1000-503X.2016.01.002

• Orginal Article • Previous Articles     Next Articles

Culture and Identification of Bone Marrow Mesenchymal Stem Cells from Enhanced Green Fluorescent Protein-transgenic Rats

Hui-juan LIU1, Ruo-yu HU2, Wang-juan DAI1, Yuan-guo LIU2, Bo MIN2, Li JIANG1()   

  1. 1Department of Pediatrics,
    2Department of Cardiothoracic Surgery,Zhongda Hospital,Southeast University,Nanjing 210009,China
  • Received:2015-04-15 Online:2016-01-15 Published:2016-03-02
  • Supported by:
    Supported by the National Natural Sciences Foundation of China(81370739) and the Natural Science Foundation of Jiangsu Province(BK20131303)

Abstract:

Objective To isolate,culture,and identify bone marrow mesenchymal stem cells(BMSCs) from enhanced green fluorescent protein (EGFP)-transgenic rats in vitro. Methods Bone marrows were isolated from tibia and femur of healthy EGFP-transgenic rats of specific pathogen free (SPF) grade. Then,the whole bone marrow adherent method was used for isolation,culture,and purification of BMSCs. The morphological change was noted by continuous observation under inverted fluorescence microscope. The growth curve of cells was drawn through the method of CCK-8 and the proliferation compared with wild type BMSCs. The surface markers of BMSCs were detected by flow cytometry. The BMSCs were induced to differentiate into osteoblasts,adipocytes,and chondrocytes lineages. The EGFP-BMSCs were transplanted into the rats intravenously,and the expression of GFP was detected. Results BMSCs stably expressing EGFP gene were obtained successfully,with the fusiform-shaped appearance and the forming of circinate cell colonies. The growth curve of EGFP-MSCs showed the characteristic of active proliferation,showing no significant difference compared with the wild-type BMSCs. The expression rates of the surface markers of BMSCs CD29,CD90,CD34,CD49d,and CD45 were 99.4%,96.4%,0.171%,0.049%,and 0.038%. The GFP were detected in lung 3 days after transplantation. After osteogenic,adipogenic,and chondrogenic induction,oil red-O and alizarin red positive signals and toluidine blue positive cells were detected. Conclusions High-purity BMSCs stably expressing green fluorescent protein gene can be cultured using the whole bone marrow adherent method. EGFP does not affect the stem cell properties and expresses stably after transplantation. The cells can be used as seed cells for subsequent research.

Key words: bone marrow mesenchymal stem cells, green fluorescent protein, induction differentiation

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