腺相关病毒介导β-神经生长因子基因转染内皮祖细胞

摘要

背景：基因疗法为许多难治性疾病的治疗带来了新思路，选择合适的细胞、基因及载体是基因治疗的关键。目的：利用重组腺相关病毒载体介导β-神经生长因子基因转染大鼠骨髓源内皮祖细胞，观察目的基因β-神经生长因子的表达情况及对内皮祖细胞增殖的影响。　　方法：分离、培养、鉴定大鼠骨髓源内皮祖细胞，使用携带β-神经生长因子基因的重组腺相关病毒(rAAV-GFP-β-NGF)转染内皮祖细胞，根据绿色荧光蛋白荧光表达情况检测转染效率。利用 ELISA法在蛋白水平检测β-神经生长因子的表达情况，MTT法检测目的基因对内皮祖细胞增殖活性的影响。　　结果与结论：成功从大鼠骨髓中分离培养出内皮祖细胞，经鉴定符合内皮祖细胞特征。β-神经生长因子基因成功转染内皮祖细胞，转染率为65.3%。基因转染组细胞培养上清中检测到β-神经生长因子蛋白的表达，且可持续10 d，空白组和空载病毒转染组未检测到β-神经生长因子的表达。重腺相关病毒转染后内皮祖细胞增殖活性增高，与空载病毒转染组和空白组相比差异有显著性意义(P<0.05)，空白组和空载病毒转染组之间增殖活性差异无显著性意义(P>0.05)。结果表明，经重组腺相关病毒介导可成功将β-神经生长因子基因转染入大鼠骨髓源内皮祖细胞，使其持续表达β-神经生长因子蛋白并促进内皮祖细胞增殖。%BACKGROUND:Nowadays, gene therapy has become a new trend for disease therapy and brought promise for some refractory diseases. Its key is to choose proper cel s, genes and vectors. OBJECTIVE:To use recombinant adeno-associated virus mediatedβ-nerve growth factor (β-NGF) to transfect rat bone marrow-derived endothelial progenitor cel s in vitro, and to investigate the effect ofβ-NGF expression on the proliferation of endothelial progenitor cel s. METHODS:The endothelial progenitor cel s were isolated, cultured and identified from the bone marrow of rats. Empty vector or recombinant adenovirus-associated virus containingβ-NGF gene was transferred into endothelial progenitor cel s. We examined the transfection efficiency by fluorescence expression of green fluorescent protein. Expression ofβ-NGF protein was detected using ELISA, and its effect on the proliferation of endothelial progenitor cel s was determined using MTT method. RESULTS AND CONCLUSION:Rat endothelial progenitor cel s were isolated and cultured successful y in vitro and were identified positive by the function of cel s and immunofluorescence staining. The endothelial progenitor cel s were infected directly by the recombinant adenovirus-associated virus containingβ-NGF gene with an efficiency of 65.3%.β-NGF protein was detected in the culture supernatant of transfected endothelial progenitor cel s, which reached a high level at 10 days after gene transfection. Furthermore, there was noβ-NGF protein in the blank and empty vector groups. After transfection, the proliferative ability of endothelial progenitor cel s was increased, which was significantly higher than the blank and empty vector groups (P<0.05). But there was no difference between the latter two groups (P>0.05). These findings suggest that recombinant adenovirus-associated virus containingβ-NGF gene can be successful y transferred into rat bone marrow-derived endothelial progenitor cel s and promote the proliferation of endothelial progenitor cel s.