Experimental Study On The Effects Of Lentivirus-mediated NGF Transfection Of BMSCs And PRP On Osteogenic Differentiation

Objective: Prove that NGF is highly expressing after lentivirus-mediated NGF transfection into bone marrow mesenchymal stem cells,and the cells’ proliferation activity was not affected after transfection,and osteogenic differentiation is promoted by the induction of platelet-rich plasma osteogenic inducers.Methods: The cultured BMSCs were divided into three groups:group A: NGF overexpression transfection group,group B: negative virus transfection group(green fluorescent empty virus),and group C:untransfected group.BMSCs were transfected with NGF overexpression recombinant lentivirus constructed by transgenic technology.After 7 days of continuous culture,use the fluorescence microscopy to observe the transfection efficiency,and detect the expression of NGF protein in cells of groups A,B,and C by Western-Blot method.Use CCK-8 method to detect the cell proliferation activity of the three groups.Osteogenic differentiation of 3 groups was induced with an osteogenic agent containing 10%activated PRP,and the normal BMSCs were induced with the osteogenic inducer without PRP as the blank control group(group D),and use alkaline phosphatase on days 5,9,and 14 respectively,use alizarin red staining to detect calcium nodule formation at the same times.Results: After transfection of rat BMSCs with NGF recombinant lentivirus in conventional DMEM low-sugar complete culture medium,no significant difference in cell proliferation activity measured by CCK-8method(differences are not statistically significant,P>0.05).After 14 days of continuous culture,the morphology of the three groups do not change significantly.Under an inverted fluorescence microscope,green fluorescence is appearing on the 3rd day after transfection,and the fluorescence expression increased significantly at 7 days.The transfection efficiency is above 90%.Western blot analysis showed that the expression level of NGF protein in the transfection group is significantly higher than the control group after 7 days of culture.After osteogenic differentiation was induced,compared with groups B,C,and D,at each time point,the alkaline phosphatase activity of cells in group A(NGF transfection combined with PRP induction group)was significantly higher(the difference was statistically significant,P<0.05),the alkaline phosphatase activity of cells in groups A,B and C was higher than group D(The difference was statistically significant,P<0.05).After 14 days of induction,alizarin red staining showed only partial calcium nodule formation in group D,while cells in groups A,B,and C induced with10% activated PRP osteogenic inducer showed bone-like tissue formation,while NGF was converted.Conclusions: Transgenic technology recombinant NGF lentivirus can successfully transfect rat BMSCs with high expression,and transfection has no significant effect on cell proliferation.The osteogenic inducer containing 10% activated PRP and overexpressed NGF can promote the osteogenic differentiation activity of BMSCs,and the overexpression of NGF combined with PRP can promote the osteogenic differentiation of BMSCs more strongly.