Effect of lentivirus-mediated silencing of P75 neurotrophin receptor gene on osteogenic differentiation of bone marrow mesenchymal stem cells in rats

Abstract

To investigate the effect of small interfering RNA (siRNA) lentivirus-mediated silencing of P75 neurotrophin receptor (P75NTR) gene on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in rats. Three lentivirus-mediated P75NTR gene siRNA sequences (P75NTR-siRNA-1, 2, 3) and negative control (NC)-siRNA were designed and transfected into the 3rd generation Sprague Dawley (SD) rat BMSCs. The cells morphological changes were observed under an inverted microscope, and the expressions of P75NTR gene and protein in cells were detected by real-time fluorescence quantitative PCR and Western blot. Then the best silencing P75NTR-siRNA for subsequent osteogenic differentiation experiments was screened out. The 3rd generation SD rat BMSCs were randomly divided into experimental group, negative control group, and blank control group (normal BMSCs). The BMSCs of negative control group and experimental group were transfected with NC-siRNA and the selected P75NTR-siRNA lentiviral vector, respectively. The cells of each group were cultured by osteogenic induction. The expressions of osteogenic related proteins [osteocalcin (OCN) and Runx related transcription factor 2 (Runx2)] were detected by Western blot; the collagen type Ⅰ expression was observed by immunohistochemical staining; the osteogenesis of BMSCs was observed by alkaline phosphatase (ALP) detection and alizarin red staining. After lentivirus-mediated P75NTR transfected into BMSCs, the expressions of P75NTR mRNA and protein significantly reduced ( P<0.05), and the best silencing P75NTR-siRNA was P75NTR-siRNA-3. After P75NTR gene was silenced, MTT test showed that the cell proliferation in the experimental group was significantly faster than those in the two control groups ( P<0.05). After osteogenic induction, the relative expressions of OCN and Runx2 proteins, collagen type Ⅰ expression, and ALP activity were significantly higher in the experimental group than in the two control groups, the differences were significant ( P<0.05). With the prolongation of osteogenic induction, the mineralized nodules in the experimental group gradually increased. Silencing the P75NTR gene with siRNA lentivirus can promote the osteogenic differentiation of rat BMSCs and provide a new idea for the treatment of bone defects.