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Objective To study the effect of TLR4 activation with LPS on BMP9-induced osteogenic differentiation of immortalized mouse embryonic fibroblasts ( iMEFs).Methods The activation of TLR4/NF-κB signaling path-way was detected by ICC.iMEFs were treated with LPS,BAY11-7082,Adnovirus GFP and BMP9.The early osteo-genic differentiation capability of iMEFs was detected by ALP staining and quantitative assay .The later osteogenic differentiation capability was detected by alizarin red S staining .The expression of later osteogenic differentiation marker gene OCN and OPN were detected by PCR and Western blot .The change of p-Smad1/5/8 was detected by Western blot.The expression of Runx2 and Dlx5 were detected by PCR and Western blot .Results LPS can effec-tively stimulate TLR4/NF-κB signaling pathway .TLR4 activation inhibited BMP 9-induced osteogenic differentiation . BMP9-induced osteogenic differentiation related gene and Smad 1/5/8 signaling activation were inhibited by TLR4 activation .The inhibition effect was partly reversed by BAY 11-7082 ( P<0.05 ) .Conclusions TLR4 activation with LPS can inhibit BMP9-induced osteogenic differentiation of iMEFs cells via NF-κB signaling pathway .
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Objective:To investigate the effects of BMP9 combined with NGF on the osteogenic differentiation of mesenchymal stem cells(MSCs).Methods:Recombinant BMP9 adenovirus was transfected into C3H10T1/2 cells.The cells were treated by GFP,NGF,BMP9 and BMP9 + NGF respectively.The expression level of COL1,RUNX2 was detected by RT-PCR and Western blot,ALP activity was examined by ALP kit 3,12,24,48 hours,3 and 7 days after treatment,respectively.Results:The ALP activity of BMP9 + NGF group was the highest among the 4 groups.The difference in the groups firstly appeared at 3 h after treatment.The highest expression level of RUNX2 and COL1 was detected in BMP9 + NGF group.Conclusion:NGF and BMP9 may synergisticly promote osteogenic differentiation at the early stage of osteogenic induction of C3H10T1/2 cells.
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Aim To study the anti-proliferation effect of resveratrol (Res)and the role of Res-induced bone morphorgenetic protein 9 (BMP9 )in this process in colon cancer cells.Methods Crystal violet staining and flow cytomtry were introduced to assay the anti-proliferation effect of Res in LoVo cells.The effect of Res on apoptosis in LoVo cells was also detected with flow cytometry.Then,RT-PCR and Western blot assay were employed to unveil the effect of Res on the ex-pression of BMP9 .The effect of BMP9 on the anti-pro-liferation of Res in LoVo cells was analyzed with crystal violet staining and flow cytometry too.Finally,the effect of Res on the expression of ALK2 and ALK3 was assayed with RT-PCR,and the inhibitor of ALK2 and ALK3 was used to figure out the possible mechanism of BMP9 on Res-induced proliferation inhibition in LoVo cells.Results Res apparently inhibited the prolifera-tion,arrested the cell cycle at S phase in LoVo and in-creased the percentage of apopotic cells in LoVo cells. Res increased the expression of mRNA and protein of BMP9 concentration dependently. Exogenous ex-pressed-BMP9 enhanced the anti-proliferation and ap-optosis inducing effects of Res in LoVo cells, but BMP9 knockdown decreased these effects of Res.Al-though Res had no apparent effect on increasing the phosphorylation of Smad1/5/8,it increased the ex-pression of ALK2 and ALK3 .Inhibition of ALK2 and ALK3 decreased the anti-proliferation effect of Res partly in LoVo cells.Conclusion Res is potent to in-hibit the proliferation of LoVo cells,Which may be mediated by up-regulating the expression of BMP9 and its receptor at least.
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Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β(TGF-β) superfamily and play critical roles in skeletal development, bone formation and stem cell differentiation.BMP9 is one of the most osteogenic BMPs, promoting osteogenesis differentiation of periodontal ligament stem cells ( PDLSCs) both in vitro and in vivo.Recently, signal transduc-tion studies have revealed that BMP-Receptor-Smads and BMP-Receptor-Mitogen activated protein kinase ( MAPK) play vital roles in BMP9 which promote PDLSCs osteogenesis differentiation.Several studies revealed that transcription factors closely associated with os-teogenesis differentiation are found in the downstream of the Smads and MAPK pathways.This review aims to summarize our current knowledge of BMP9-mediated osteogenesis by presenting recently completed works which may help us to further elucidate these path-ways.
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Aim To investigate the role of COX-2 in BMP9 induced osteogenic differentiation,and the pos-sible mechanism underlying this function of COX-2. Methods We introduced real-time PCR, Western blot, and immunocytochemical staing to detect the effect of BMP9 on COX-2 expression.We employed chemiluminescence technique to assay ALP activities, RT-PCR to detect the expression of Smad6 and Smad7 , and Western blot to measure the expression of Runx2, Dlx-5,total Smad1/5/8,and phosphorylated Smad1/5/8.Finally,BMPR-Smad luciferase reporter assay was applied to measure the activation of BMPs/Smads signaling.Results BMP9 could induce the expression of COX-2 in C3H10T1/2 cells.Either inhibiting enzy-matic activity of COX-2 or knockdown of the expression of COX-2 reduced the BMP9 induced ALP activities in C3H10T1/2 cells,and COX-2 knockdown also inhibited the ectopic bone formation induced by BMP9 in C3H10T1/2 cells.Moreover,COX-2 knockdown inhibi-ted BMPR-Smad reporter activities and the phosphoryl-ation of Smad1/5/8,so did the expression of Smad6 and Smad7 .Conclusion COX-2 may play an impor-tant role in BMP9 induced osteogenic differentiation in MSCs by regulating the BMPs/Smads signaling trans-duction.
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In order to validate and estimate the capability of BMP9 to induce osteogenic differentiation of multipotent stem cells, three multipotent stem cells(C3H10, MEFs and BMSC) were used as target cells, and BMP9 was introduced into these cells by using recombinant adenoviruses assay, the effect of BMP9 on osteogenic differentiation of multipotent stem cells was demonstrated by using luciferase reporter assay, alkaline phosphatase(ALP) quantitative assay, calcium deposition assay, real time PCR, animal experiment and histological staining assay.The results demonstrated that BMP9 can induce ALP expression of C3H10, MEFs and BMSC by a dose dependent manner.BMP9 can also stimulate calcium deposition of C3H10 and MEFs in vitro, the osteogenic markers(ALP, Runx2, osteopontin, osteocalcin) were increased after stimulated by BMP9.BMP9 can activate canonical TGF?-Smad pathway, and promote the expression of osteogenic master gene Runx2.The animal experiment and histological staining assay show that BMP9 can induce ectopic bone formation in naked mice.To sum up, BMP9 is a more powerful cell factor to induce osteogenic differentiation of multipotent stem cells.
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Objective:ToexploreinfluenceofBMP9inmyocardiocytesdifferentiationof stem cell Invitro.Methods:TheC3H10T1/2stem cell were transfected with 2.67 ml/L pAdEasy-BMP9 Ad-GFP plasmid for 1 week and 2 weeks .The specific transcription factors of cardiomyocytes were detected in stem cells during differentiation with plasmid transfection by RT-PCR and the specific proteins in cardiomy-ocytes were detected by laser confocal microsopy after 1 week;the change of cells ultramicro structure were detected by electron micro-scope after 2 weeks.Results:Cells volume are increscent obviously,cells trend become unanimous,the connection between cells are compact,refractivity of cells enhance conspicuously after induction for 1 week.The expression of NKx 2.5,GATA-4,MEF2C could be de-tected in C3H10T1/2 stem cells with pAdEasy-BMP9 Ad-GFP plasmid tranfection.Cx43 and cTnT can be detected with plasmid tranfec-tion.Cardiomyocytes ultramicro structure can be detected after induction for 2 weeks.Conclusion:BMP9 makes a very important influ-ence in C3H10T1/2 stem cells targeted differentiation into cardiomyocytes。