关键词: P75神经生长因子受体, 神经生长因子, 过表达质粒, 慢病毒, 脂质体, 双基因转染, 骨髓间充质干细胞, 国家自然科学基金

Abstract:

BACKGROUND: As a receptor for nerve growth factor (NGF), P75 neurotrophin receptor (P75^NTR) can mediate distinct signaling pathways in different cells, but the regulatory effect in bone marrow mesenchymal stem cells has not been studied yet. Liposome and lentivirus-mediated single-gene transfection cell technology have matured, but the differences in mediating double gene transfection have not been reported.
OBJECTIVE: To construct the overexpression plasmid and lentiviral vector of rat P75^NTR and NGF, and to compare the effect and practicability of transfected bone marrow mesenchymal stem cells by lipofection and lentivirus infection in order to select the best experimental solution according to different experimental requirements in the future. 
METHODS: The gene primers of rat P75^NTR and NGF were designed. Genomic DNA was extracted for PCR amplification, and GV358-P75^NTR and GV492-NGF overexpression plasmids were constructed by recombinant plasmid vector, transfected into 293T cells, and ultracentrifuged by lentiviral vector to collect target genes. The lentivirus was expressed and the titer of the virus sample was determined. The rat primary bone marrow mesenchymal stem cells were cultured in vitro, and one group was co-transfected with plasmid GV358-P75^NTR and GV492-NGF with liposome Lipo3000, and the other group was treated with lentivirus. Negative control group and blank control group were set at the same time. The transfected bone marrow mesenchymal stem cells were routinely cultured. The expression of red and green fluorescence was observed under fluorescence microscope on days 3, 5, 7, 9, and 12, and the expression of P75^NTR and NGF proteins was detected by western blot. Bone marrow mesenchymal stem cells were digested and subcultured on day 7 after transfection. The expression of red and green fluorescence was observed by fluorescence microscope on days 3, 5, 7, 9, and 12, and the expression of P75^NTR and NGF proteins was detected by western blot. 
RESULTS AND CONCLUSION: The GV358-P75^NTR and GV492-NGF overexpression plasmids and lentivirus were constructed in accordance with the experimental design. For the abundance of red-green fluorescence in the microscope field, the liposome co-transfection group increased at first and then decreased; the lentivirus co-transfection group continued to increase; and the lentivirus co-transfection group was higher than the liposome co-transfection group at each time point. In the liposome co-transfection group, the fluorescence expression of the subcultured cells after transfection was lower than that of the non-subcultured cells, and the lentivirus co-transfection group showed no changes. The expression of P75^NTR and NGF proteins in the lentiviral infection group was significantly higher than that in the liposome group at each time point. The expression levels of P75^NTR and NGF proteins in the two target gene transfection groups were significantly higher than those in the negative control group and blank group. Both P75^NTR and NGF genes could be transfected into rat bone marrow mesenchymal stem cells via liposome and lentivirus co-transfection. Compared with lentivirus-mediated double gene transfection, liposome-mediated double gene transfection is a relatively simple method that has lower dependency to experimental equipment and lower cost. However, the infection efficiency is significantly lower than that of lentivirus-mediated double gene transfection. The target gene continues to express after lentivirus-mediated double gene transfection, and no gene loss occurs after subculture.

Key words: P75 neurotrophin receptor, nerve growth factor, overexpression plasmid, lentivirus, liposome, double gene transfection, bone marrow mesenchymal stem cells, National Natural Science Foundation of China