人β-NGF及BDNF基因共转染对大鼠骨髓基质细胞分化的影响  

Effects of human IS-nerve growth factor and brain-derived neural neurotrophic factor gene cotransfection on the differentiation of rat bone marrow stromal stem cells

作　　者：王凡[1] 毛伯镛[2] 陈俊杰[3] 贺民[2] 刘艳辉[2] 鞠延[2] 

机构地区：[1]成都市第三人民医院神经外科,610031 [2]四川大学华西医院神经外科,成都610041 [3]四川大学基础医学与法医学院生物化学研究所重组DNA室,成都610041

出　　处：《中华神经医学杂志》2009年第10期977-981,共5页Chinese Journal of Neuromedicine

摘　　要：目的探讨人β-神经生长因子（β-NGF）和脑源性神经营养因子（BDNF）基因共转染对大鼠骨髓基质细胞（BMSCs）分化的影响。方法梯度离心法分离培养大鼠胫股骨骨髓中BMSCs，用Src癌基因同源区3抗体（SH3）的免疫细胞化学染色鉴定BMSCs。按照3μL脂质体／1μg（3μL）pSVCEP NGF／BDNF-CAT质粒的比例共转染第一代BMSCs，并转染pEGFP—C1质粒作为转染的标记。BMSCs培养4周后，荧光显微镜观察增强型绿色荧光蛋白（EGFP）的表达并计算转染率，细胞免疫荧光染色后用激光扫描共聚焦显微镜观察表达产物以及BMSCs分化情况。结果体外培养能够获得纯化的原代和传代BMSCs，传代后仍然保持增殖和分化功能；免疫组化SH3染色阳性证实为纯化的BMSCs。人β-NGF／BDNF基因共转染BMSCs后，BMSCs能够稳定和持续表达NGF和BDNF；BMSCs也能表达Nestin、NSE、NF—M和GFAP。结论经脂质体介导的外源性目的基因NGF／BDNF cDNA均能分别和共同在BMSCs中成功表达，基因转染后的BMSCs能诱导分化为神经前体细胞或神经样细胞。Objective To observe the effect of human β-nerve growth factor （β-NGF） and brain-derived neurotrophic factor （BDNF） gene co-transfection on the differentiation of rat bone marrow stromal stem cells （BMSCs）. Methods BMSCs were isolated from the femoral and tibial bone marrow of SD rats by density-gradient centrifugation and identified using immunocytochemistry of Src homology 3 （SH3）. The first-passage BMSCs were transfected with pSVCEP-NGF-CAT and pSVCEP-BDNF-CAT plasmids via liposome, and also with pEGFP-C1 as the marker of transfection. After a 4-week cell culture, the expression of EGFP in the transfected cells was detected using fluorescence microscope, and the transfection efficiency was calculated. Laser scanning confocal microscope was employed to observe the expressions of the neural precursor cell markers NGF, BDNF, nestin, neuron specific enolase （NSE）, NF and glial fibrillary acidic protein （GFAP） and evaluate the differentiation of the transfected cells. Results The primary and passaged BMSCs with high purity were obtained successfully without any decrease in the proliferation and differentiation potentials. The long spindle-shaped cells in the early passages displayed high proliferative activity, and immtmocytochemistry of SH3 identified the purified cells as the bona fide BMSCs. NGF and BDNF genes were stably expressed in the BMSCs after the transfection, and the transfected cells were also positive for nestin, NSE, NF-M and GFAP. Conclusions Effective ectopic expression of human β-NGF and BDNF genes is achieved in rat BMSCs after liposome-mediated gene transfection. The BMSCs are capable of differentiating into neuronal precursor cells that express several neural proteins and resemble the immature neurons or glial cells after induction with neurotrophic factors.

关 键 词：骨髓基质细胞 神经生长因子 脑源性神经营养因子 脂质体转染 细胞分化 

分 类 号：R686[医药卫生—骨科学]