慢病毒-血管内皮生长因子165载体的构建及其在脂肪组织来源的干细胞中过表达的研究

Construction of VEGF165 lentiviral vector and over-expression in adipose tissue-derived stem cells

目的构建慢病毒-血管内皮生长因子。（VEGF165）载体并转染脂肪组织来源干细胞（ADSCs）以得到用于基因治疗的种子细胞。方法通过聚合酶链反应（PCR）方法将EHSl001—68950485313912克隆突变成VEGF165（NM_001025368）转录本后采用三质粒系统（pGC—FU、pHelpl．0和pHelp2．0）进行包装慢病毒。通过定时定量PCR测定滴度后使用慢病毒-VEGFl65转染ADSCs。免疫荧光、Westernblot鉴定VEGF165在ADSCs中的表达。结果PCR（535bp）、DNA测序、Westernblot（49×10^3）检测证明成功构建的载体为VEGF165与绿色荧光蛋白（GFP）融合表达载体。实时定量PCR确定病毒滴度达到2×10^8TU／ml。慢病毒-VEGF165贷载体转染ADSCs后经免疫荧光验证约95％ADSCs可表达VEGF165，同时Westernblot也证明基因稳定表达。结论通过获得稳定表达VEGF165的ADSCs，为血管性疾病特别是糖尿病性勃起障碍的治疗奠定了基础。

Objective Many studies found that stem cells can act as gene therapeutic vector. But there is few related report focus on the Adipose tissue-derived stem ceils （ADSCs）. Therefore we intend to construct a lentiviral vascular endothelial growth factor165 （ VEGF165 ） over expression vector and infect the ADSCs to produce therapeutic seed cells. Methods VEGF165 transcript （NM_001025368） fragment was origin from the EHS1001-6895048 5313912 clone and mutated to the purpose transcript by polymerase chain reaction （PCR） method. The Lentivirus was enveloped with 3 plasmids （pGC-FU, pHelper 1.0 and pHelper 2. 0）. And then use the successful constructed vectors infected the ADSCs （MOI = 20） after titer determination. Stable expression of VEGF16s in ADSCs was confirmed by immunofluorescence （95% affected） staining and western blotting analysis. Results VEGFI65 was linked to the green fluorescent protein （GFP） fused vector which was verified by the PCR （535 bp）, DNA sequencing, Western blotting （49 × 10^3）. Quantitative PCR determined that the virus titer is up to 2 × 10^8 which. VEGF165 transduced cells could show green fluorescence which was confirmed by immunofluorescence. And the Western blotting analyses confirmed VEGF165 expression in VEGF165-transduced cells. Conclusion We obtained the VEGF165 over-expression ADSCs and these cells could use as a gene therapeutic tool and may be applied for vascular regeneration, especially in treatment of erectile dysfunction.