Studies On Telomerase Reverse Transcriptase Gene Modification Of Ovis Aries Bone Marrow-derived Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMSCs) are multipotent stem cells in bone marrow stroma,with a superior proliferative capacity in vitro and could be induced to differentiate into a variety of cells under certain induction conditions,such as bone, cartilage, adipose cells which are derived from the mesoderm,nerve cells are derived from the ectodermal et al.BMSCs have became the ideal seed cells of tissue engineering,because of convenience and weak immunogenicity,had the important research significance and clinical application value in cell replacement therapy, gene therapy and tissue regeneration-However, BMSCs proliferation in vitro and the capacity of multi-directional differentiation will decline gradually with the continuous passage, which limit the effect in the field of research and application.Utilizing telomerase reverse transcriptase (TERT) gene to modify BMSCs,could make it become "immortalized" cells of a long-term sustained proliferation, "immortalization" BMSCs could be used as standard cell in cell engineering and tissue engineering research sphere,and play a more important role in the basic research and in the field of medicine.In this paper,we have used the BMSCs of sheep as the object of study,constructed the eukaryotic expression vector of the TERT gene transfected into the BMSCs.We got the BMSCs cell lines stably expressed TERT gene after selection, by making analyses of the biological characteristics and identification of multiple induced differentiation,which proved that TERT gene modified BMSCs could proliferate normally and efficiently in vitro, and still keep the ability of multi-directional differentiation.Our main results showed as following,1.Getting sheep TERT gene cDNA sequence in vitro clone,gene recombination with the pcDNA3.1-EGFP vector, which successfully constructed the TERT eukaryotic expression vector.2.The TERT eukaryotic expression vector by liposome transfection, G418 screening, RT-PCR detection,we got the BMSCs cell lines stably expressed TERT gene after selection.3.The growth curve showed that BMSCs were modified by TERT gene,which had more stronger ability of proliferation in vitro than BMSCs without genetic modification.TERT- BMSCs cells expressed stem cell surface factors Oct4, Sox2 and Nanog.TERT-BMSCs were induced into adipogenic cells and nerve cells,the differentiated cells not only appeared the peculiar lipid droplets of adipogenic cells,the specific morphology and the Nisslbody of nerve cells,but also the expression of adipogenic cells specific genes PPAR and LEPTIN,nerve cells specific genes NSE and GFAP.showed that TERT-BMSCs cells still maintain the ability of multi-directional differentiation potential.The research established the necessary foundation for exploring the mechanism of BMSCs cell immortalization.