Lentivirus-mediated Silencing Of P75NTR Combined With NGF Overexpression And Transfection Of BMSCs Combined With Demineralized Bone Matrix For Heterotopic Osteogenesis

Objective: To investigate the effects of P75 neurotrophin receptor(P75NTR)combined with nerve growth factor(NGF)overexpression on the proliferation activity of bone marrow mesenchymal stem cells(BMSCs)and the ability of DBM to construct tissue engineered bone heterotectopia.The novel composite gene of NGF and P75 NTR is of great significance in the study of tissue engineering,and provide theoretical basis and new ideas for the treatment of bone defects.Methods: BMSCs of SD rats were cultured and passaged by adherent isolation method,flow cytometry detects stem cell surface markers.The third generation BMSCs were transfected with lentivirus mediated P75 NTR gene silencing(group B),NGF overexpression gene(Group C),P75 NTR silencing and NGF overexpression double genes(Group D),respectively,and untransfected cells as control(Group A).After 7 days of transfection,the expression of fluorescent protein of the target gene was observed by fluorescence microscope.cell counting kit 8 method was used to detect the activity of cells for 8 days after transfection.the expression of P75 NTR and NGF protein in each group was detected by Western blot.The adhesion of BMSCs to demineralized bone matrix(DBM)was observed by inverted phase contrast microscope and scanning electron microscope after transfection of P75 NTR silencing and NGF overexpression double genes.After transfection,BMSCs and DBM were co-cultured to prepare four groups of tissue-engineered bone,which were respectively placed in the dorsal subcutaneous tissue of 8-week-old SD rats to construct subcutaneous ectopic osteogenesis model(n = 6).HE staining was performed at 4 and 8 weeks after operation.ALP staining was used to observe the formation of calcium nodules at 8weeks after operation.The expressions of Runt-related transcription factor 2(Runx2),alkaline phosphatase(ALP),and osteocalcin(OCN)were detected by real time fluorescent quantitative PCR.Results: The third-generation BMSCs were long fusiform or spindle-shaped.After cell morphology observation and flow cytometry identification of CD14,CD29,CD34,CD44,and CD45,the cultured cells were BMSCs.At 7 days after transfection,there was no fluorescence expression in group A,red fluorescence expression in group B,green fluorescence expression in group C,and red-green composite fluorescence expression in group D.The fluorescence expression rate of target gene was about 70%.Western blot detection showed that the relative expression of P75 NTR protein in groups A and C was significantly higher than that in groups B and D,and the relative expression of NGF protein in groups C and D was significantly higher than that in groups A and B(P<0.05).With the passage of time,the cell proliferation activity increased in all groups,especially in group D,which was significantly higher than that in group A at 3-8 days(P<0.05).The results of inverted phase contrast microscope and scanning electron microscope showed that BMSCs could adhere well to DBM.In the subcutaneous ectopic osteogenesis experiment,HE staining showed that at 4and 8 weeks after operation,the more bone tissue was formed in group D than in the other 3 groups.ALP staining showed that group D had the highest ALP activity and better osteogenic expression.Compared with group A,the relative expressions of Runx2,ALP,and OCN m RNAs in group D were significantly higher than those in group A(P<0.05).Conclusion: Silencing P75 NTR and NGF overexpression double genes co-transfected BMSCs with DBM to construct tissue engineered bone has good ectopic osteogenic ability.By increasing NGF level and closing P75 NTR apoptosis channel,it can not only improve cell activity,but also promote bone tissue regeneration.