重组质粒pcDNA3-β-NGF的构建及其转染小鼠骨髓间充质干细胞生物学活性的研究

Biological characteristics of bone marrow mesenchymal stem cells from GFP transgenic mice transfected with human β-nerve growth factor gene

目的:构建人神经生长因子β亚基(β-NGF)真核表达载体,转染荧光小鼠骨髓间充质干细胞(MSCs)并研究其生物学活性。方法:全骨髓贴壁培养法分离、培养和纯化荧光小鼠MSCs,构建真核表达载体pcDNA3-β-NGF,并转染MSCs;免疫组织化学染色检测β-NGF的表达,观察β-NGF阳性的MSCs对小鼠海马神经元生长的作用。结果:荧光小鼠MSCs可发出明亮的绿色荧光,且荧光不随培养时间延长和传代次数增加而衰减。重组质粒pcDNA3-β-NGF转染MSCs后β-NGF阳性率为(37.12±2.14)%,高于MSCs组[(2.36±0.62)%]和空白对照组[(1.43±0.76)%],差异有统计学意义(P<0.05)。pcDNA3-β-NGF转染MSCs上清液培养的新生小鼠海马神经元的突起长度[(31±3)μm]较pcDNA3转染MSCs组[(23±4)μm]明显增加,差异有统计学意义(P<0.05),表明β-NGF基因转染MSCs培养上清液中的产物能促进小鼠海马神经元的突起生长,具有明显的生物学活性。结论:成功构建了pcDNA3-β-NGF真核表达载体,其转染的荧光小鼠MSCs能正确、稳定表达和分泌有生物学活性的β-NGF。

AIM: To investigate the changes of biological characteristics of GFP transgenic mouse bone marrow mesenchymal stem cells( MSCs),which were transfected with human β-nerve growth factor( β-NGF) gene in a recombinant eukaryotic expression vector. METHODS: MSCs obtained from GFP transgenic mice were isolated,cultured and purified by the whole bone marrow adherence methods. Human β-NGF gene was transfected into the MSCs by a recombinant eukaryotic expression vector. The β-NGF expression in the MSCs was detected by the method of immunocytochemistry.Hippocampal neurons from neonatal mice were cultured with culture supernatant of the MSCs transfected with pcDNA3-β-NGF and the biological characteristics of the MSCs were investigated 3 d after culture under inverted phase-contrast microscope. RESULTS: The β-NGF positive rate of MSCs in pcDNA3-β-NGF transfection group [( 37. 12 ± 2. 14) %]was significantly higher than that in MSCs control group [( 2. 36 ± 0. 62) %] and blank control group [( 1. 43 ± 0. 76) %]. The neurite length of neonatal mouse hippocampal neurons cultured with culture supernatant from pcDNA3-β-NGF-transfected MSCs [( 31 ± 3) μm] was significantly longer than that in negative control group [( 23 ± 4) μm],suggesting that MSCs transfected with β-NGF gene maintained better biological characteristics. CONCLUSION: The constructed recombinant eukaryotic expression vector of human β-NGF gene can be transfected into MSCs efficiently and NGF can be effectively expressed in MSCs. MSCs transfected with β-NGF gene are capable of stable expression and secretion of β-NGF,and maintenance of better biological characteristics.