Coexpressing EGFP and rat Ang-1 gene of recombinate lentivirus transfects to the cultured rat bone marrow-derived mesenchymal stem cells in vitro
AIM:To observe coexpressing enhanced green fluorescent protein (EGFP) and rat angiopoietin-1 (Ang-1) gene of recombinate lentivirus transfers to the cultured rat bone marrow-derived mesenchymal stem cells (rMSC) in vitro. METHODS: To package the compressing EGFP and Ang-1 gene of recombinate lentivirus in vitro. Then the virus titer was examined. The rMSCs were infected by obtained lentiviral particles, the expression of EGFP and the transfection efficiency were examined under fluorescent microscope after transfection. The relative quantitative expression of Ang-1 mRNA and protein were examined at different times by real-time fluorescence quantitative PCR (real-time qPCR)and Western blot. Human umbilical vein endothelial cells (HUVEC) were co-cultured respectively with the supernatant fluid of transfected rMSCs (Ang-1-rMSCs)/rMSCs which had been cultured for 48 h, MTT was used to determine the OD value of HUVECs. RESULTS: The titer of lentiviral vector particles was found to be 6.1 × 1010 TU/L. The result of the infection in rMSCs showed that the transduction efficiency in Ang-1-rMSCs was (92.7 ± 3.01)% after 5 d; real-time qPCR and Western Blot showed Ang-1 expression had been in the peak during transduction 3 - 7 d, after then it was in slow downtrend. MTT methods showed the proliferative capacity of HUVECs was better in the co-culture system of Ang-1-rMSCs groups than that of rMSCs groups (P ＜ 0.01). CONCLUSION: The recombinate lentivirus can transfer into rMSCs cultured in vitro, in which Ang-1 expression had been in the peak during transduction 3 - 7 d and the biological effect of Ang-1 gene had been.
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维普
