Over-expression of VEGF165 in the adipose tissue-derived stem cells via the lentiviral vector
Over-expression of VEGF165 in the adipose tissue-derived stem cells via the lentiviral vector
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通过慢病毒载体在脂肪组织干细胞中过表达 VEGF165
DOI：
10.3760/cma.j.issn.0366-6999.2011.19.026
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发表时间：
2011-10-05
影响因子：
6.1
通讯作者：
Deng Chun-hua
中科院分区：
文献类型：
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作者：
Sun Xiang-zhou;Liu Gui-hua;Deng Chun-hua
背景许多研究者研究了干细胞作为基因治疗载体的可能性。但目前关于脂肪组织来源的干细胞（ADSCs）的研究报道较少。方法采用PCR方法对EHS 1001 -68950485313912克隆进行突变，产生VEGF（165）基因的保守片段（NM_001025368）。用pGC FU、pHelper 1.0和pHelper 2.0质粒在293 T细胞中包被慢病毒。然后用滴度测定后的载体转染ADSC（感染复数=20）。免疫荧光染色、酶联免疫吸附试验（ELISA）和Western blotting分析证实VEGF 165在ADSCs中稳定表达。定量PCR检测病毒滴度达2 × 10 ~（8）。经免疫荧光染色证实，VEGF 165转导的细胞可显示绿色荧光（几乎95%）。ELISA法可检测到VEGF的浓度为850.86- 1202.13pg/ml（平均（923.00 +/- 31.22）pg/ml），但在GFP转导的细胞中未检测到Westernblotting分析也证实了VEGF 165在转染细胞中的表达。表达ADSC的细胞可以用作细胞治疗工具，并且可以应用于血管再生，特别是在勃起功能障碍的治疗中。中华医学杂志2011;124（19）：3093-3097
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 VEGF165 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 VEGF165 in ADSCs was confirmed by immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis.Results DNA sequencing and 293T transfection verified VEGF165 was linked to the GFP fused vector. The virus titer is up to 2x10(8) determined by quantitative PCR. VEGF165 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 VEGF165 expression in VEGF165-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. Chin Med J 2011;124(19):3093-3097