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

Abstract

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 immunocytochemistry 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 tmnsfection. 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. Key words: Bone marrow stromal stem cells; Nerve growth factor; Brain-derived neurotrophic factor; Liposome-mediated transfection; Cell differentiation