BACKGROUND: Many researchers studied the possibility of using stem cells as gene therapeutic vector. But few related reports on the adipose tissue-derived stem cells (ADSCs) are available. Therefore we intended to construct a lentiviral VEGF(165) expression vector and then infect the ADSCs to produce therapeutic seed cells. METHODS: EHS1001-68950485313912 clone was mutated by PCR method to produce consensus fragment of VEGF(165) transcript (NM_001025368). Lentivirus was enveloped with pGC-FU, pHelper 1.0 and pHelper 2.0 plasmids in 293T cells. And then the ADSCs (multiplicity of infection = 20) were transfected with the vectors after titer determination. Stable expression of VEGF(165) in ADSCs was confirmed by immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis. RESULTS: DNA sequencing and 293T transfection verified VEGF(165) was linked to the GFP fused vector. The virus titer is up to 2 × 10(8) determined by quantitative PCR. VEGF(165) transduced cells could show green fluorescence confirmed by immunofluorescence staining (almost 95%). ELISA analyses could detect out the density of VEGF was 850.86 - 1202.13 pg/ml (mean (923.00 ± 31.22) pg/ml) in the supernatant of VEGF(165)-transduced cells but not detected in the GFP-transduced cells (P < 0.001) and the Western blotting analyses also confirmed VEGF(165) expression in VEGF(165)-transduced cells. CONCLUSIONS: The VEGF(165) over-expression ADSCs were obtained and may be used as a cell therapeutic tool and may be applied for vascular regeneration, especially in the treatment of erectile dysfunction.
BACKGROUND: Many researchers studied the possibility of using stem cells as gene therapeutic vector. But few related reports on the adipose tissue-derived stem cells (ADSCs) are available. Therefore we intended to construct a lentiviral VEGF(165) expression vector and then infect the ADSCs to produce therapeutic seed cells. METHODS: EHS1001-68950485313912 clone was mutated by PCR method to produce consensus fragment of VEGF(165) transcript (NM_001025368). Lentivirus was enveloped with pGC-FU, pHelper 1.0 and pHelper 2.0 plasmids in 293T cells. And then the ADSCs (multiplicity of infection = 20) were transfected with the vectors after titer determination. Stable expression of VEGF(165) in ADSCs was confirmed by immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis. RESULTS: DNA sequencing and 293T transfection verified VEGF(165) was linked to the GFP fused vector. The virus titer is up to 2 × 10(8) determined by quantitative PCR. VEGF(165) transduced cells could show green fluorescence confirmed by immunofluorescence staining (almost 95%). ELISA analyses could detect out the density of VEGF was 850.86 - 1202.13 pg/ml (mean (923.00 ± 31.22) pg/ml) in the supernatant of VEGF(165)-transduced cells but not detected in the GFP-transduced cells (P < 0.001) and the Western blotting analyses also confirmed VEGF(165) expression in VEGF(165)-transduced cells. CONCLUSIONS: The VEGF(165) over-expression ADSCs were obtained and may be used as a cell therapeutic tool and may be applied for vascular regeneration, especially in the treatment of erectile dysfunction.