Effect Of Lentivirus-mediated P75NTR Gene Silencing On Osteogenic Differentiation Of Rat BMSCs

Objective:Objective: To investigate the small interfering RNA(si RNA)lentivirus-mediated silencing of P75 nerve growth factor receptor(P75NTR)gene transfected into rat BMSCs to induce osteogenic differentiation and culture,and to study the silencing through a series of observation experiments The effect of P75 NTR gene on the growth state and osteogenic differentiation of rat bone marrow mesenchymal stem cells(BMSCs).Methods:First design three lentiviral-mediated P75 NTR gene si RNA sequences(P75NTR-si RNA-1,2,3)and negative control(NC)-si RNA,respectively,transfect the third generation SD rat BMSCs;ordinary optics Observe cell morphology changes under an inverted phase contrast microscope,real-time fluorescent quantitative Polymerase Chain Reaction(PCR)to detect P75 NTR gene expression,and Western blot to detect cell P75 NTR protein expression.Through the above experiment,the P75NTR-si RNA-3 with the best silencing effect was selected for subsequent experiments.Then,the third-generation rat BMSCs were randomly divided into experimental group,negative control group and blank control group.Among them,the negative control group and experimental group were transfected with NC-si RNA and the selected P75NTR-si RNA lentiviral vector to transfect BMSCs.The control group was normal BMSCs.The cell proliferation after transfection was detected by MTT method;after osteogenic differentiation and culture of each group of cells,the expression of osteocalcin(OCN)and Runx related transcription factor 2(Runx2)were detected by Western blot.Histochemical staining was used to observe the expression of type I collagen,ALP and Alizarin Red staining were used to observe the osteogenesis.Results:After lentivirus-mediated P75 NTR transfected into BMSCs,the expressions of P75 NTR m RNA and protein significantly reduced(P<0.05),and the best silencing P75NTR-si RNA was P75NTR-si RNA-3.After P75 NTR gene 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.Conclusion:Silencing the P75 NTR gene with si RNA lentivirus can promote the osteogenic differentiation of rat bone marrow mesenchymal stem cells and provide a new idea for the treatment of bone defects.