The Effect And Mesenchymal Research Of FGF2Regulate BMP9-induced Osteogenic Differentiation Of Mesenchymal Stem Cells

Objective:Analysis the effects of osteogenic differentiation and relatedmolecular mechanism of FGF2on BMP9induced mesenchymal stem cells(mesenchymal stem cells, MSCS).Methods:Firstly,detected the effect of exogenous FGF2or BMP9onendogenous BMP9or FGF2protein expression in MSCs by westernblot;then C3H10T1/2cells were infected by Ad-RFP and AdR-FGF2withdifferent concentration,at the same time prepared the BMP9conditionedmedium,added the conditioned medium into C3H10T1/2cells when theAd-RFP and AdR-FGF2infected after24h,the early maker Alkalinephosphotase(ALP) activity of osteogenic differentiation were detected at5dand7d;the effect of FGF2and BMP9on proliferation of C3H10T1/2cellswere detected by crystal violet staining;then detected the late maker calciumdeposition with FGF2and BMP9induced osteogenic differentiation byAlizarin Red S staining;the change of osteogenic differentiation late makerOPN and OCN were detected with FGF2and BMP9induced on C3H10T1/2cells by western blot.Then,we detected the change of BMP9-induced osteogenic diffe rentiation related target genes(ID1、 ID2、 ID3) and key transcriptionfactor Runx2on C3H10T1/2cells by PCR,detected the protein change ofRunx2by western blot;detected the change of classic signaling pathwayBMP9-Smad1/5/8by luciferase reporter assay and western blot,observed thechange of non-classic signaling pathway MAPK-p38and MAPK-erk1/2bywestern blot;detected the change of ALK1and ALK2of BMP9Ⅰtypereceptor were detected by PCR.Lastly,C3H10T1/2co-infected by Ad-RFP/Ad-BMP9、Ad-FGF2/Ad-BMP9、 Ad-F GF2、Ad-BMP9were subcutaneously injectedto BALB/c nude mice respectively to investigate the ectopic osteogenesis ofFGF2on BMP9-induced osteogenic differentiation,and bone tissue sectionswere stained with H&E,Alcian Blue,Masson’s Trichome,observed the bonematurity.Results: The results showed no any effect of the protein expressionbetween exogenous FGF2or BMP9on endogenous BMP9or FGF2;BMP9induced ALP activity decreased gradually with increased concentration ofFGF2infection;the crystal violet staining displayed FGF2and BMP9have acertain role promoting on MSCs proliferation,and the effect is more obviouswhen both are used;Another kind of mesenchymal stem cell from Mouseembryonic fibroblasts(MEFs),FGF2inhibited the ALP activity of MEF cellsby BMP9-induced;the late maker calcium deposition of BMP9-inducedosteogenic differentiation were inhibited by FGF2on C3H10T1/2cells and MEF cells;the protein expression of OPN and OCN were inhibited by FGF2on C3H10T1/2cells.The PCR results showed BMP9-induced osteogenic differentiationrelated target genes(ID1、 ID2、ID3) and key transcription factor Runx2were inhibited by FGF2on C3H10T1/2cells,and the protein of Runx2wasinhibited by FGF2;FGF2and BMP9can activate MAPK-p38andMAPK-erk1/2signaling pathway,but,it’s no effect that FGF2on theactivation of MAPK by BMP9-induced on C3H10T1/2cells;notably,theactivation of BMP9-induced classic Smad1/5/8signaling pathway wereinhibited by FGF2,showed BMP9-induced SBE luciferase activity andphosphorylation of Smad1/5/8were inhibited by FGF2;the PCR resultsshowed FGF2inhibited the expression of BMP9-induced osteogenic relatedreceptors ALK1and ALK2.The ectopic bone formation in subcutaneously displayed that BALB/cnude mice were subcutaneously injected on C3H10T1/2cells co-infected byAd-RFP/Ad-BMP9Ad-FGF2/Ad-BMP9,BMP9-induced bone formationwere inhibited by FGF2,and bone maturity were inhibited by section stainingshowed.Conclusions:FGF2suppressed BMP9-induced the osteogenicdifferentiation and bone formation on mesenchymal stem cells, mainly realized through inhibited the classic BMPs-Smad1/5/8signaling pathwayinduced by BMP9into osteogenic differentiation.