Lentivirus-GFP/Lentivirus-mCherry Transfection of HUVECs

Lentivirus-GFP/Lentivirus-mCherry (Heyuan Biotechnology Co., Ltd., China) was used to transfect HUVECs with green fluorescent (GFP) and red fluorescent mCherry protein markers, respectively. HUVECs were grown in 24-well plates at a density of 7 × 10⁴ cells/well, then added with 500 μl RPMI-1640 complete growth medium and cultured at 37°C, in 5% CO₂ incubator (Heraeus Company, Germany) for 24 h. Next, the culture medium was replaced with DMEM high-glucose complete medium (Gibco BRL, United States) with 5 μg/ml polybrene (Heyuan Biotechnology Co., Ltd., China) and preconfigured virus solution with MOI = 40. Polybrene is a cationic polymer, which can neutralize the electric charge to promote binding between the lentivirus and cell membrane. After 72 h of infection, the infection efficiency was evaluated by a fluorescence microscope (Olympus, Japan); 0.5 μg/ml purinomycin was selected to maintain HUVECs labeled with green fluorescent protein (GFP-HUVECs) or red fluorescent protein (mCherry-HUVECs). In the following experiments, GFP-HUVECs were used to characterize the migration of adjacent VECs, and mCherry-HUVECs to indicate the adhesion of circulating VECs.