| Abstract Objective:This study was aimed to clone VCAM-1cDNA gene and to construct retrovirus vector PMSCVmigr-1-VCAM-1, explored the effect of over-expression of VCAM-1gene in mesenchymal stem cell(MSC) on the immunological capacity of MSC to provide a basis for optimizing clinical use of MSC.Methods:The cDNA encoding mouse VCAM-1was construced into the recombinant retrovirus vector MIGR1which expressed the report gene EGFP by the DNA recombination. The reconstructed plasmid MIGR1-VCAM-1, empty plasmid MIGR1and packaging plasmid ECOS were transfected into T293cell lines by Lipofectamine, respectively, and then the supernatant from T293cells were infected mouse MSCs cell line C3H10T1/2. The transfective efficiency was determined by inverted fluorescence microscope and flow cytometry. VCAM-1expression on MSC was evaluated by FACS. Moreover, the inhibitory effect of VCAM-1-MSC on lymphocytic transformation was tested by3H-TdR incorporation assay.Results:The recombinant retro viral vector MIGR1-VCAM-1was successful construcedand mouse VCAM-1has been stably overexpressed in MSC cell line C3H10T1/2. Furthermore, the morphology of overexpression VCAM-1MSC cell line (C3H10T1/2-MlGRl-VCAM-1/MSC) was no change. Comparing with the MSC transfected with empty vector (C3H10T1/2-MIGR1), MSC over-expressing VCAM-1could significantly suppress T lymphocytes proliferation in vitro by nonspecific mitogens stimulation in a dose-dependent manner. When MSC:T cells was1:10, the cpm value of C3H10T1/2-MIGR1-VCAM-1/MSC was significant decreased by65.3%, while the vector transfected MSC yield a decrease of29.8%. The inhibitory efficiency of VCAM-1high MSC was2.16fold higher than that of the vector transfected control.Conclusion:Overexpression of VCAM-1could enhance the immunosupression effect in T cells activation and proliferation. These results provide new insight of the experimental and the theoretical basement for cell therapy in immune diesease by MSC containing over expression VC AM-1. |
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