腺相关病毒介导β-神经生长因子基因转染内皮祖细胞

Transfection of adeno-associated virus encoding beta-nerve growth factor into endothelial progenitor cells

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(r AAV-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 cells, genes and vectors. OBJECTIVE： To use recombinant adeno-associated virus mediated β-nerve growth factor（β-NGF） to transfect rat bone marrow-derived endothelial progenitor cells in vitro, and to investigate the effect of β-NGF expression on the proliferation of endothelial progenitor cells. METHODS： The endothelial progenitor cells were isolated, cultured and identified from the bone marrow of rats. Empty vector or recombinant adenovirus-associated virus containing β-NGF gene was transferred into endothelial progenitor cells. We examined the transfection efficiency by fluorescence expression of green fluorescent protein. Expression of β-NGF protein was detected using ELISA, and its effect on the proliferation of endothelial progenitor cells was determined using MTT method. RESULTS AND CONCLUSION： Rat endothelial progenitor cells were isolated and cultured successfully in vitro and were identified positive by the function of cells and immunofluorescence staining. The endothelial progenitor cells were infected directly by the recombinant adenovirus-associated virus containing β-NGF gene with an efficiency of 65.3%. β-NGF protein was detected in the culture supernatant of transfected endothelial progenitor cells, which reached a high level at 10 days after gene transfection. Furthermore, there was no β-NGF protein in the blank and empty vector groups. After transfection, the proliferative ability of endothelial progenitor cells was increased, which was significantly higher than the blank and empty vector groups（P〈0.05）. But there was nodifference between the latter two groups（P〉0.05）. These findings suggest that recombinant adenovirus-associated virus containing β-NGF gene can be successfully transferred into rat bone marrow-derived endothelial progenitor cells and promote the proliferation of endothelial progenitor cells.