| Objective:To research the influence of inducible membrane protein combined with extract of Salvia miltiorrhiza on osteogenic differentiation of rat BMSCs,and to explore the relationship between inducible membrane protein and extract of Salvia miltiorrhiza on osteogenic differentiation of rat BMSCs and HIF-1αsignaling pathwayMethods:The experiment was divided into blank group,salvia miltiorrhiza extract group(experimental group 1),induced membrane protein group(experimental group 2),induced membrane protein combined with salvia miltiorrhiza extract group(experimental group 3),and rat BMSCs were cultured in different media.In blank group,cells were cultivated in complete medium.Experimental group 1:Cells were cultured in complete medium containing 0.8mg/mL Salvia miltiorrhiza extract.Experimental group 2:Cells were cultured in a complete medium containing 100μg/ml of induced membrane protein.Experimental group 3:Cells were cultivated in a complete medium including 100μg/ml induced membrane protein and 0.8mg/ml extract of Salvia miltiorrhiza.BMSCs were cultured for 1 week without using the above methods,and cell proliferation was detected by CCK-8.The morphological variation of cells each of the experimental groups were detected by inverted microscope.With alizarin red staining cells each test mineralized nodule formation and selected three view to count.PCR detection of each cell osteogenesis markers(Runx-2,Col1,OCN)gene expression level.By western blot experiments,we test the protein expression level of osteogenesis markers(Runx-2,Col1,OCN).Western blot experiments testing groups HIF-1α and downstream factor(iN-OS,TGF-beta,VEGF)protein expression level.Result:CCK-8 test indicated that contrast to the blank group,the cells in the salvia miltiorrhiza extract group,induced membrane protein group,induced membrane protein combined with salvia miltiorrhiza extract group had no significant effect on cell proliferation after 24h,48h and 72h culture(P>0.05).② the cell morphological changes,as can be seen under inverted microscope,blank cells are long spindle,salvia miltiorrhiza extract group,the induced membrane proteins,membrane proteins induced joint salvia miltiorrhiza extract group of short cells into a fusiform or spindle,some of which present scaly or polygon,it can be seen that cells treated by salvia miltiorrhiza extract,induced membrane protein changed in form,the preliminary direction differentiation of BMSCs have asked.③ On the basis of the alizarin red staining pictures and counting,contrast to the blank group,the number of mineralized nodules in the salvia miltiorrhiza extract group,induced membrane protein group,induced membrane protein combined with salvia miltiorrhiza extract group was significantly increased;Compared with the salvia miltiorrhiza extract group,the cell mineralization nodules in the induced membrane protein group and the induced membrane protein combined with salvia miltiorrhiza extract group were significantly increased.Compared with induced membrane protein group,cells in induced membrane protein combined with Salvia miltiorrhiza extract group showed significantly more mineralized nodules.④PCR detection showed that contrast to the blank group,the expression levels of cell osteogenic markers(RUNX-2,COL1,OCN)genes in the extract group,induced membrane protein group and induced membrane protein were increased,with statistical significance(P<0.05).Compared with the salvia miltiorrhiza extract group,the expression levels of cell osteogenesis markers(RUNX-2,COL1,OCN)genes in the inductive membrane protein group and the inductive membrane protein combined with salvia miltiorrhiza extract group increased,with statistical significance(P<0.05).Compared with the induced membrane protein group,the expression levels of cell osteogenesis markers(RUNX-2,COL1,OCN)genes in the induced membrane protein combined with Salvia miltiorrhiza extract group were increased,with statistical significance(P<0.05).⑤ Western blot showed that contrast to blank group,the protein expression levels of cell osteogenesis markers(Runx-2,Col1,OCN)in Salvia miltiorrhiza extract group,induced membrane protein group,induced membrane protein combined with Salvia miltiorrhiza extract group were increased,with statistical significance(P<0.05).Compared with the salvia miltiorrhiza extract group,the expression levels of cell osteogenesis markers(RUNX-2,COL1,OCN)protein in the inductive membrane protein group and the inductive membrane protein combined with salvia miltiorrhiza extract group were increased,with statistical significance(P<0.05).Compared with the induced membrane protein group,the expression levels of cell osteogenesis markers(RUNX-2,COL1,OCN)protein in the induced membrane protein combined with Salvia miltiorrhiza extract group were increased,with statistical significance(P<0.05).⑥Western blot analysis showed that,contrast to blank group,the expression levels of cell HIF-1α and downstream factors(iN-OS,TGF-β,VEGF)of salvia miltiorrhiza extract group,induced membrane protein group,induced membrane protein contrast to salvia miltiorrhiza extract group were increased,with statistical significance(P<0.05).Contrasted to Salvia miltiorrhiza extract group,the expression levels of cellular HIF-1α and downstream factors(IN-OS,TGF-β,VEGF)induced membrane protein group and induced membrane protein contrasted to Salvia miltiorrhiza extract group were increased,and the difference was statistically significant(P<0.05).Contrasted to the membrane protein induced group,the protein expression levels of cell HIF-1α and downstream factors(iN-OS,TGF-β,VEGF)of induced membrane protein contrasted to Salvia miltiorrhiza extract group were increased,and the difference was statistically significant(P<0.05).Conclusion:① Salvia miltiorrhiza extract can accelerate osteogenic transdifferentiation of BMSCs cells.②Induced membrane protein can promote osteogenic transdifferentiation of rat BMSCs cells.③Compared with Salvia miltiorrhiza extract,induced membrane protein promoted osteogenic transdifferentiation of rat BMSCs cells more strongly④Compared with extract and induced membrane protein,the osteogenic transdifferentiation of BMSCs cultured with induced membrane protein combined with extract of Salvia miltiorrhiza was stronger.⑤ Salvia miltiorrhiza extract and inducible membrane protein promote osteogenic transdifferentiation of BMSCs,which may be related to the activation of HIF-1A signaling pathway. |
