title: Over-expression of VEGF165 in the adipose tissue-derived stem cells via the lentiviral vector.
abstract: 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.
authors: Department of Urology, First Affiliated Hospital of Sun Yet-sen University, Guangzhou, Guangdong 510080, China. sunxzh08@hotmail.com
authors: Xiang-Zhou
authors: XZ
authors: Sun
authors: Gui-Hua
authors: GH
authors: Liu
authors: Zhuo-Qing
authors: ZQ
authors: Wang
authors: Fu-Fu
authors: FF
authors: Zheng
authors: Jun
authors: J
authors: Bian
authors: Yan-Ping
authors: YP
authors: Huang
authors: Yong
authors: Y
authors: Gao
authors: Ya-Dong
authors: YD
authors: Zhang
authors: Chun-Hua
authors: CH
authors: Deng
mesh_terms: D000273:Adipose Tissue / Q000166:cytology*; D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D016086:Lentivirus / Q000235:genetics*; D051381:Rats; D013234:Stem Cells / Q000737:chemistry*; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis*
chemical_list: C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A