Abstract:

[Abstract]   Objective   To investigate the optimal condition of lentivirus，which was recombined with marker gene of
enhanced green fluorescent protein (Lentivirus-EGFP) transfect human umbilical cord wharton’s jelly-derived mesenchymal stem cells (HUWMSCs) and the effect of transfection on the proliferation in HUWMSCs.  Methods   HUWMSCs were transfected with EGFP by lentivirus vector in vitro via different multiplicity of transfection (MOI) in four different transfection methods (A, B, C and D). The fluorescence expression and the transfection efficiency in different methods were analyzed by both fluorescent microscope and flow cytometry. The proliferation rates of infected HUWMSCs was evaluated by MTT method.  Results   The transfection efficiency was10.6%-87.3% after4days in all experimental groups, which showed the dose-effect relationship with MOI. Polybrene (5mg/L) could significantly increase the transfection efficiency (P＜0.05). Results of MTT assay showed that there were significant differences in the proliferation rates of infected HUWMSCs between different transfection methods (P<0.001). There was better cell proliferation in method A (MOI=10) group and method B (MOI=10) group than that of other groups.   Conclusion   Method B (MOI=10) is the optimal transfection method in this experiment. HUWMSCs could be transfected by lentivirus-EGFP with high efficiency and could stably express transfected gene within2weeks, which is a safe and effective gene transfer vector.

Key words: mesenchymal stem cells, flow cytometry, lentivirus, genetic vectors, gene therapy, transfection, cell proliferation