Overexpression of P75 neurotrophin receptor negatively
regulates osteogenic differentiation of bone marrow mesenchymal stem cells in vitro

doi:10.3969/j.issn.2095-4344.1856

Abstract

Abstract:

BACKGROUND: P75 neurotrophin receptor (P75^NTR) is widely expressed in nerve tissues and cells, and plays a dual role in promoting or inhibiting differentiation. P75^NTR is also overexpressed in local tissues with fracture nonunion. Therefore, P75^NTR is studied for the osteogenic differentiation of bone marrow mesenchymal stem cells, which is of great significance to provide important targets for the clinical treatment of fracture nonunion.

OBJECTIVE: To elucidate the effect of P75^NTR overexpression on osteogenic differentiation of rat bone marrow mesenchymal stem cells in vitro.

METHODS: The bilateral femurs of Sprague-Dawley rats were selected, and the rat bone marrow mesenchymal stem cells were extracted by whole bone marrow separation and adherence method and subcultured in vitro. The P75^NTR overexpression plasmid GV358-P75^NTR expressing enhanced green fluorescent protein was constructed, and the P75^NTR overexpression lentiviral vector was collected by empty lentiviral vector packaging. Rat bone marrow mesenchymal stem cells primary cultured in vitro for 10 days were selected, and seeded into culture plates after digestion. P75^NTR overexpression lentivirus and related infection reagents were added for subsequent infection experiments. After 7 days of infection, the expression of green fluorescent protein was observed by fluorescence microscope and overexpression of P75^NTR protein was detected by western blot. Transfected cells were cultured for 7 days in a conventional medium, followed by culture in the osteogenic differentiation medium. Alkaline phosphatase activity was quantified by enzyme linked immunosorbent assay on the 7^th, 10^th, and 14^th days after osteogenic induction. Formation of mineralized nodules was observed by alizarin red staining on the 7^th and 14^th days after osteogenic induction.

RESULTS AND CONCLUSION: P75^NTR overexpression lentiviral vector-infected bone marrow mesenchymal stem cells expressed green fluorescent protein (infection efficiency was about 90%), and the expression of P75^NTR protein was significantly increased, which was significantly different from that in the negative control group (P < 0.05). Cell model of P75^NTR overexpression was successfully constructed. Compared with the negative control and blank control groups, the alkaline phosphatase activity of the P75^NTR overexpression group was significantly decreased at the corresponding time point after osteogenic induction, the number of mineralized nodules was significantly reduced, and cell aggregation and distribution were significantly weakened (P < 0.05). To conclude, P75^NTR overexpression negatively regulates the osteogenic differentiation of rat bone marrow mesenchymal stem cells cultured in vitro. Overexpression of P75^NTR in local tissues inhibits the osteogenic differentiation of surrounding bone marrow mesenchymal stem cells, which may be an important factor for bone defects or fracture nonunion.

Key words: P75 neurotrophin receptor, bone marrow mesenchymal stem cells, osteogenic differentiation, lentivirus, alkaline phosphatase, National Natural Science Foundation of China

CLC Number:

Zhu Lunjing, Duan Jiangtao, Huang Yijie, Wang Ning, Chen Junyi, Ma Yuegang, Bei Chaoyong. Overexpression of P75 neurotrophin receptor negatively regulates osteogenic differentiation of bone marrow mesenchymal stem cells in vitro[J]. Chinese Journal of Tissue Engineering Research, 2020, 24(1): 20-26.

Figures/Tables 7

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