Lentivirus-mediated P75 neurotrophin receptor silencing combined with nerve growth factor overexpression promotes proliferation of rat bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2099

Abstract

Abstract:

BACKGROUND: P75 Neurotrophin receptor (P75^NTR) is one of the receptors for nerve growth factor (NGF). P75^NTR plays a dual role in promoting proliferation or apoptosis in various cell tissues, and is highly expressed at fracture nonunion sites. However, excessive NGF can shut down P75^NTR receptor, thereby saving damaged cells. Therefore, the study regarding co-transfection of silenced P75^NTR and NGF overexpression is of great significance for the proliferation of bone marrow mesenchymal stem cells and provides new ideas for clinical treatment of fracture nonunion.

OBJECTIVE: To observe the effect of lentivirus-mediated silencing of P75^NTR combined with NGF overexpression on the proliferation of bone marrow mesenchymal stem cells in Sprague-Dawely rats.

METHODS: Bone marrow mesenchymal stem cells from Sprague-Dawley rats were cultured to the third generation in vitro and were divided into blank control group, negative control group, silent p75^NTR group, NGF overexpression group, and silent p75^NTR combined with NGF overexpression group. Lentivirus-mediated silencing of P75^NTR and overexpression of NGF were transfected into rat bone marrow mesenchymal stem cells to induce P75^NTR silencing and NGF overexpression. Inverted fluorescence microscopy was used to observe changes in cell morphology on day 3 after transfection. Flow cytometry was used to detect transfection efficiency and western blot method was applied to detect the expression of P75^NTR and NGF. Finally, the cell proliferation activity was detected by MTT method and cell counting kit-8 method.

RESULTS AND CONCLUSION: Cell growth and distribution were good after co-transfection of lentivirus. The transfection efficiency of the double-gene lentiviral vector exceeded 70%. Compared with the blank control and negative control groups, the expression of P75^NTR protein was significantly down-regulated, and the expression of NGF was profoundly up-regulated in the silent p75^NTR combined with NGF overexpression group. Compared with the blank control and negative control groups, cell proliferation was significantly increased in the other three groups (P < 0.05), and the fastest proliferation was observed in the silent p75^NTR combined with NGF overexpression group. To conclude, silencing P75^NTR combined with NGF overexpression co-transfection can promote the proliferation of bone marrow mesenchymal stem cells from Sprague-Dawley rats.

Key words:

bone marrow mesenchymal stem cells, lentivirus, P75 nerve growth factor receptor, nerve growth factor, transfection, cell proliferation

CLC Number:

Wang Ning, Chen Junyi, Zhu Lunjing, Duan Jiangtao, Wang Ye, Li Zhijun, Bei Chaoyong.

Lentivirus-mediated P75 neurotrophin receptor silencing combined with nerve growth factor overexpression promotes proliferation of rat bone marrow mesenchymal stem cells [J]. Chinese Journal of Tissue Engineering Research, 2020, 24(25): 3988-3993.

Figures/Tables 9

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