【摘要】 目的研究真核表达载体pcDNA3．1／V5-His-FGF2(成纤维细胞生长因子2)在骨髓间充质干细胞(MSCs)中的表达及其对MSCs的影响。方法基因克隆构建pcDNA3．1／V5-His-FGF2真核表达载体,利用FuGENETM6转染大鼠MSCs,RT-PCR、免疫荧光检测其表达情况,四甲基偶氮唑蓝(MTT)检测其对MSCs生物学活性的影响。结果构建了含FGF2基因的真核表达载体;RT-PCR证实外源FGF2基因可转染到MSCs;免疫荧光检测转染24 h即可在MSCs中表达;MTT法检测转染48、72 h后细胞吸光度值(0．3871±0．0433、0．4349±0．0319)明显高于空载体组(0．3457±0．0162、0．3881±0．0434)(t1=2．833、t2=2．313,P<0．05)和未转染组(0．3378±0．0215、0．3641±0．0365)(t1=3．227、t2=3．650,P<0．05),而空载体对细胞增殖无影响(P> 0．05)。结论构建了FGF2真核表达质粒,并在MSCs中成功表达,为FGF2基因治疗的研究奠定了坚实的基础。更多还原

【Abstract】 Objective To construct eukaryotic vector containing fibroblast growth factor 2(FGF2) gene and to study the expression in mesenchymal stem cells(MSCs) from rat bone marrow. Methods Plasmid pcDNA3.1/V5-His containing rat FGF2 gene was constructed and then transfected into the primary cultured rat MSCs under the mediation of FuGENETM6. The expression was verified by using RT-PCR and immunofluorescence staining. The proliferation of the transfected MSCs was evaluated by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) method. Results Eukaryotic vector containing FGF2 gene was constructed, RT-PCR identified that FGF2 gene had been transfected into mesenchymal stem cells. Immunofluorescence staining of post-transfected MSCs was positive at 24 h after transfection. MTT method showed that the proliferation of FGF2-transfected MSCs (0.3871±0.0433,0.4349±0.0319) were increased significantly compared with control vector group (0.3457±0.0162, 0.3881±0.0434)(t1 = 2.833,t2 = 2.313,P< 0.05),non-transfected group (0.3378±0.0215,0.3641±0.0365)(t1 = 3.227,t2 = 3.650,P < 0.05) at 48 and 72 h post-transfection,respectively. Conclusions The construction of eukaryotic expression plasmid carrying FGF2 and its expression in MSCs provides a basis of gene therapy using FGF2 gene.更多还原