Department of Urology, First Affiliated Hospital of Sun Yet-sen University, Guangzhou, Guangdong 510080, China.
Chin Med J (Engl). 2011 Oct;124(19):3093-7.
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.
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.
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.
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.
许多研究人员研究了将干细胞用作基因治疗载体的可能性。但是，关于脂肪组织来源的干细胞（ADSCs）的相关报道很少。因此，我们旨在构建慢病毒 VEGF（165）表达载体，然后感染 ADSCs 以产生治疗性种子细胞。
通过 PCR 方法突变 EHS1001-68950485313912 克隆，产生 VEGF（165）转录物（NM_001025368）的共识片段。慢病毒在 293T 细胞中用 pGC-FU、pHelper 1.0 和 pHelper 2.0 质粒包裹。然后在测定滴度后用载体转染 ADSCs（感染复数= 20）。通过免疫荧光染色、酶联免疫吸附测定（ELISA）和 Western blot 分析证实 VEGF（165）在 ADSCs 中的稳定表达。
DNA 测序和 293T 转染证实 VEGF（165）与 GFP 融合载体相连。病毒滴度通过定量 PCR 测定高达 2×10^8。通过免疫荧光染色（几乎 95%）证实转导细胞可显示绿色荧光，可检测到 VEGF（165）转导细胞上清液中 VEGF 的密度为 850.86-1202.13 pg/ml（平均值（923.00±31.22）pg/ml），而 GFP 转导细胞中未检测到（P<0.001），Western blot 分析也证实了 VEGF（165）在 VEGF（165）转导细胞中的表达。
获得了 VEGF（165）过表达的 ADSCs，可作为细胞治疗工具，可用于血管再生，特别是在治疗勃起功能障碍方面。
