Pulp loss is accompanied by the functional impairment of defense,sensory,and nutrition supply.The approach based on endogenous stem cells is a potential strategy for pulp regeneration.However,endogenous stem cell sour...Pulp loss is accompanied by the functional impairment of defense,sensory,and nutrition supply.The approach based on endogenous stem cells is a potential strategy for pulp regeneration.However,endogenous stem cell sources,exogenous regenerative signals,and neovascularization are major difficulties for pulp regeneration based on endogenous stem cells.Therefore,the purpose of our research is to seek an effective cytokines delivery strategy and bioactive materials to reestablish an ideal regenerative microenvironment for pulp regeneration.In in vitro study,we investigated the effects of Wnt3a,transforming growth factor-beta 1,and bone morphogenetic protein 7(BMP7)on human dental pulp stem cells(h-DPSCs)and human umbilical vein endothelial cells.2D and 3D culture systems based on collagen gel,matrigel,and gelatin methacryloyl were fabricated to evaluate the morphology and viability of h-DPSCs.In in vivo study,an ectopic nude mouse model and an in situ beagle dog model were established to investigate the possibility of pulp regeneration by implanting collagen gel loading BMP7.We concluded that BMP7promoted the migration and odontogenic differentiation of h-DPSCs and vessel formation.Collagen gel maintained the cell adhesion,cell spreading,and cell viability of h-DPSCs in 2D or 3D culture.The transplantation of collagen gel loading BMP7 induced vascularized pulp-like tissue regeneration in vivo.The injectable approach based on collagen gel loading BMP7 might exert promising therapeutic application in endogenous pulp regeneration.展开更多
Objective To evaluate the effects of retinoic acid(RA)on expression of bone morphogenetic protein 7(BMP-7)in rat fetus with cleft palate,and the effects of RA on proliferation and apoptosis of osteoblasts.Methods All-...Objective To evaluate the effects of retinoic acid(RA)on expression of bone morphogenetic protein 7(BMP-7)in rat fetus with cleft palate,and the effects of RA on proliferation and apoptosis of osteoblasts.Methods All-trans RA(ATRA)was used to induce congenital cleft palate in Wistar rat.BMP-7 mRNA expression in maxillary bone tissue of fetal rats was measured by Northern blotting analysis.Flow cytometry and MTT assay were used to measure the apoptosis and proliferation of ATRA-treated MC-3T3-E1 cells.BMP-7 mRNA and protein expressions in ATRA-treated MC-3T3-E1 cells were detected by RT-PCR and Western blotting analysis.Results ATRA could induce cleft palate of rat fetus.The incidence rate of cleft palate induced by 100 mg/kg ATRA(45.5%)was significantly higher than 50 mg/kg ATRA(12.5%,P<0.05).BMP-7 mRNA expression decreased in maxillary bone tissue of rat fetus with cleft palate.MC-3T3-E1 cells proliferation treated with 1×10-6 mol/LATRA decreased by 60%,the cell apoptosis increased by 2 times.BMP-7 mRNA and protein levels in MC-3T3-E1 cells treated with 1×10-6 mol/L ATRA decreased by 60% and 80%,respectively,compared with ATRA-untreated cells(P<0.05).Conclusions BMP-7 may play an important role in embryonic palate development.RA may possess the ability to down-regulate cell proliferation through regulation of BMP-7 gene expression.展开更多
To construct the recombinant adeno-associated virus (rAAV) vector with human bone morphogenetic protein 7 (BMP7) and observe the BMP7 mRNA expression in vitro, BMP7 CDS se-quence was cloned into expression plasmid pAA...To construct the recombinant adeno-associated virus (rAAV) vector with human bone morphogenetic protein 7 (BMP7) and observe the BMP7 mRNA expression in vitro, BMP7 CDS se-quence was cloned into expression plasmid pAAV-MCS of AAV Helper Free System. The recombi-nant plasmid was identified with enzyme digestion and sequencing. The recombinant plasmid, pAAV-RC, pHelper were co-transfected into AAV-293 cells according to the calcium phosphate-based protocol. The viral stock was collected by 4 rounds of freeze/thaw. After purified and concentrated, the recombinant virus titer was determined by dot-blot assay. HEK293 cells were transfected with the recombinant virus at different MOI, and the expression of BMP7 mRNA was detected by RT-PCR. The results showed rAAV-BMP7 was constructed and packaged successfully. The physical particle titer was 2.5×1011 vector genomes/mL. There was different expression level of BMP7 mRNA after transfecton. These data suggested that recombinant AAV mediated a stable expression of hBMP7 mRNA in 293 cells. The AAV production method may pave the way of an effective strategy for the jaw bone defection around dental implants.展开更多
BACKGROUND:Some researches demonstrate that exogenous bone morphogenetic protein 7(BMP-7) can protect ischemic cerebral nerve tissue and promote recovery of motor energy function;however,there is lack of direct eviden...BACKGROUND:Some researches demonstrate that exogenous bone morphogenetic protein 7(BMP-7) can protect ischemic cerebral nerve tissue and promote recovery of motor energy function;however,there is lack of direct evidences of endogenous BMP-7 effect. OBJECTIVE:To observe the expression of endogenous BMP-7 in nerve tissue with ischemic-hypoxic injury and investigate the possible effects on damaged nerve tissue. DESIGN:Observational contrast animal study. SETTING:Department of Anatomy and Histoembryology,Peking University Health Science Center. MATERIALS:The experiment was carried out in the Nerve Researching Laboratory of Anatomy Department,Peking University Health Science Center from October 2006 to March 2007. A total of 25 adult male SD rats weighing 250-300 g and several newborn SD rats were selected from Experimental Animal Center,Peking University Health Science Center. Rabbit-anti-BMP-7 polyclonal antibody was provided by Wuhan Boster Company. METHODS:① Adult rats were randomly divided into ischemia group(n =10),sham operation group(n = 10) and normal group(n =5). Right external-internal carotid artery occlusion was used to infarct middle cerebral artery of adult rats in the ischemia group so as to copy focal cerebral infarction models. Line cork was inserted in crotch of internal and external carotid artery of adult rats in the sham operation group,while adult rats in the normal group were not given any treatments. ② Cerebral cortex of newborn rats was separated to obtain cell suspension. Cells which were cultured for 10 days were divided into control group and hypoxia/reoxygenation group. And then,cells in the hypoxia/reoxygenation group were cultured in hypoxic incubator for 4 hours and given reoxygenation for 24 hours. MAIN OUTCOME MEASURES:Immunohistochemical method was used to measure expression of BMP-7 in cerebral cortex at 24 hours after ischemia/reperfusion culture and in primary hypoxic culture. RESULTS:① At 24 hours after cerebral ischemia,expression of BMP-7 in cerebral cortex on ischemic side was stronger than that on non-ischemic side in adult rats;meanwhile,numbers of cell expression were increased. However,expression of BMP-7 was not detected in bilateral cerebral cortex of adult rats in both control group and sham operation group. ② After hypoxia of cerebral cortex in primary culture,positive products of BMP-7 were observed in plasma of neuron,but expression of BMP-7 was not found in normal cerebral cortex. CONCLUSION:Endogenous BMP-7 has protective effects on nerve tissue induced by ischemic-hypoxic injury.展开更多
Mesenchymal stem cells(MSCs)originate from many sources,including the bone marrow and adipose tissue,and differentiate into various cell types,such as osteoblasts and adipocytes.Recent studies on MSCs have revealed th...Mesenchymal stem cells(MSCs)originate from many sources,including the bone marrow and adipose tissue,and differentiate into various cell types,such as osteoblasts and adipocytes.Recent studies on MSCs have revealed that many transcription factors and signaling pathways control osteogenic development.Osteogenesis is the process by which new bones are formed;it also aids in bone remodeling.Wnt/β-catenin and bone morphogenetic protein(BMP)signaling pathways are involved in many cellular processes and considered to be essential for life.Wnt/β-catenin and BMPs are important for bone formation in mammalian development and various regulatory activities in the body.Recent studies have indicated that these two signaling pathways contribute to osteogenic differen-tiation.Active Wnt signaling pathway promotes osteogenesis by activating the downstream targets of the BMP signaling pathway.Here,we briefly review the molecular processes underlying the crosstalk between these two pathways and explain their participation in osteogenic differentiation,emphasizing the canonical pathways.This review also discusses the crosstalk mechanisms of Wnt/BMP signaling with Notch-and extracellular-regulated kinases in osteogenic differentiation and bone development.展开更多
AIM: To investigate the antifibrotic effects of bone morphogenetic protein-7 (BMP-7) on Schistosoma japonicum (S. japonicum )-induced hepatic fibrosis in BALB/C mice. METHODS: Sixty BALB/C mice were randomly divided i...AIM: To investigate the antifibrotic effects of bone morphogenetic protein-7 (BMP-7) on Schistosoma japonicum (S. japonicum )-induced hepatic fibrosis in BALB/C mice. METHODS: Sixty BALB/C mice were randomly divided into three groups, including a control group (group A, n = 20), model group (group B, n = 20) and BMP-7 treated group (group C, n = 20). The mice in group B and group C were abdominally infected with S. japonicum cercariae to induce a schistosomal hepatic fibrosis model. The mice in group C were administered human recombinant BMP-7. Liver samples were extracted from mice sacrificed at 9 and 15 wk after modeling. Hepatic histopathological changes were assessed using Masson's staining. Transforming growth factor-beta 1 (TGF-β1), alpha-smooth muscle actin (α-SMA), phosphorylated Smad2/3 (pSmad2/3) and Smad7 protein levels and localization were measured by Western blotting and immunohistochemistry, respectively, and their mRNA expressions were detected by reverse transcriptionpolymerase chain reaction (RT-PCR). RESULTS: The schistosomal hepatic fibrosis mouse model was successfully established, as the livers of mice in group B and group C showed varying degrees of typical schistosomal hepatopathologic changes such as egg granuloma and collagen deposition. The degree of collagen deposition in group C was higher than that in group A (week 9: 22.95±6.66vs 2.02±0.76; week 15: 12.84±4.36 vs 1.74±0.80; P<0.05), but significantly lower than that in group B (week 9: 22.95±6.66 vs 34.43±6.96; week 15: 12.84±4.36 vs 18.90±5.07;P<0.05) at both time points. According to immunohistochemistry data, the expressions of α-SMA, TGF-β1 and pSmad2/3 protein in group C were higher than those in group A (α-SMA: week 9: 21.24±5.73 vs 0.33±0.20; week 15: 12.42±4.88 vs 0.34±0.27; TGF-β1: week 9: 37.00±13.74 vs 3.73±2.14; week 15: 16.71±9.80 vs 3.08±2.35; pSmad2/3: week 9: 12.92±4.81 vs 0.83±0.48; week 15: 7.87±4.09 vs 0.90±0.45; P<0.05), but significantly lower than those in group B (α-SMA: week 9: 21.24±5.73 vs 34.39±5.74; week 15: 12.42±4.88 vs 25.90±7.01; TGF-β1: week 9: 37.00±13.74 vs 55.66±14.88; week 15: 16.71±9.80 vs 37.10±12.51; pSmad2/3: week 9: 12.92±4.81 vs 19.41±6.87; week 15: 7.87±4.09vs 13.00±4.98;P<0.05) at both time points; the expression of Smad7 protein in group B was higher than that in group A and group C at week 9 (8.46±3.95 vs 1.00±0.40 and 8.46±3.95 vs 0.77±0.42; P<0.05), while there were no differences in Smad7 expression between the three groups at week 15 (1.09±0.38 vs 0.97±0.42 vs 0.89±0.39; P>0.05). Although minor discrepancies were observed, the results of RT-PCR and Western blotting were mainly consistentwith the immunohistochemical results. CONCLUSION: Exogenous BMP-7 significantly decreased the degree of hepatic fibrosis in both the acute and chronic stages of hepato-schistosomiasis, and the regulatory mechanism may involve the TGF-β/Smad signaling pathway.展开更多
BACKGROUND Liver fibrosis is a refractory disease whose persistence can eventually induce cirrhosis or even liver cancer.Early liver fibrosis is reversible by intervention.As a member of the transforming growth factor...BACKGROUND Liver fibrosis is a refractory disease whose persistence can eventually induce cirrhosis or even liver cancer.Early liver fibrosis is reversible by intervention.As a member of the transforming growth factor-beta(TGF-β)superfamily,bone morphogenetic protein 7(BMP7)has anti-liver fibrosis functions.However,little is known about BMP7 expression changes and its potential regulatory mechanism as well as the relationship between BMP7 and TGF-βduring liver fibrosis.In addition,the mechanism underlying the anti-liver fibrosis function of BMP7 needs to be further explored.AIM To investigate changes in the dynamic expression of BMP7 during liver fibrosis,interactions between BMP7 and TGF-β1,and possible mechanisms underlying the anti-liver fibrosis function of BMP7.METHODS Changes in BMP7 expression during liver fibrosis and the interaction between BMP7 and TGF-β1 in mice were observed.Exogenous BMP7 was used to treat mouse primary hepatic stellate cells(HSCs)to observe its effect on activation,migration,and proliferation of HSCs and explore the possible mechanism underlying the anti-liver fibrosis function of BMP7.Mice with liver fibrosis received exogenous BMP7 intervention to observe improvement of liver fibrosis by using Masson’s trichrome staining and detecting the expression of the HSC activation indicator alpha-smooth muscle actin(α-SMA)and the collagen formation associated protein type I collagen(Col I).Changes in the dynamic expression of BMP7 during liver fibrosis in the human body were further observed.RESULTS In the process of liver fibrosis induced by carbon tetrachloride(CCl4)in mice,BMP7 protein expression first increased,followed by a decrease;there was a similar trend in the human body.This process was accompanied by a sustained increase in TGF-β1 protein expression.In vitro experiment results showed that TGF-β1 inhibited BMP7 expression in a time-and dose-dependent manner.In contrast,high doses of exogenous BMP7 inhibited TGF-β1-induced activation,migration,and proliferation of HSCs;this inhibitory effect was associated with upregulation of pSmad1/5/8 and downregulation of phosphorylation of Smad3 and p38 by BMP7.In vivo experiment results showed that exogenous BMP7 improved liver fibrosis in mice.CONCLUSION During liver fibrosis,BMP7 protein expression first increases and then decreases.This changing trend is associated with inhibition of BMP7 expression by sustained upregulation of TGF-β1 in a time-and dose-dependent manner.Exogenous BMP7 could selectively regulate TGF-β/Smad pathway-associated factors to inhibit activation,migration,and proliferation of HSCs and exert antiliver fibrosis functions.Exogenous BMP7 has the potential to be used as an antiliver fibrosis drug.展开更多
AIM:To observe the effect of Danshao Huaxian capsule(DHC)on the expression of Gremlin and bone morphogenetic protein-7(BMP-7)in the liver of hepatic fibrosis rats.METHODS:A total of 75 male Wistar rats were randomly d...AIM:To observe the effect of Danshao Huaxian capsule(DHC)on the expression of Gremlin and bone morphogenetic protein-7(BMP-7)in the liver of hepatic fibrosis rats.METHODS:A total of 75 male Wistar rats were randomly divided into a normal control group(A),a CCl4-induced hepatic fibrosis model group(B),a natural recovery group(C),a low-dose DHC-treated group(D),and a high-dose DHC-treated group(E),with 15 rats in each group.Liver fibrosis was induced by subcutaneous injections of carbon tetrachloride(CCl4)and a highlipid/low-protein diet for 8 wk,except for the rats in group A.Then,the rats in the two DHC-treated groups were administered 0.5 and 1.0 g/kg DHC by gastrogavage once per day for 8 successive weeks,respectively.By the end of the experiment,the level of transforming growth factorβ1(TGF-β1)in the liver homogenate was determined by an enzyme-linked immunosorbent assay.The mRNA and protein expression of Gremlin and BMP-7 in the liver tissue was determined by reversetranscription polymerase chain reaction,an immunohistochemical assay,and Western blot analysis.RESULTS:Compared with group A,the level of TGF-β1and the mRNA and protein expression of Gremlin were significantly higher in group B(TGF-β1:736.30±24.40μg/g vs 284.20±18.32μg/g,P<0.01;mRNA of Gremlin:80.40±5.46 vs 49.83±4.20,P<0.01;positive protein expression rate of Gremlin:38.46%±1.70%vs 3.83%±0.88%,P<0.01;relative protein expression of Gremlin:2.81±0.24 vs 0.24±0.06,P<0.01),and the mRNA and protein expression of BMP-7was significantly lower in group B(mRNA:54.00±4.34vs 93.99±7.03,P<0.01;positive protein expression rate:28.97%±3.14%vs 58.29%±6.02,P<0.01;relative protein expression:0.48±0.31 vs 1.05±0.12,P<0.01).Compared with groups B and C,the degree of hepatic fibrosis was significantly improved,and the level of TGF-β1 and the mRNA and protein expression of Gremlin were significantly lowered in the two DHCtreated groups(TGF-β1:523.14±21.29μg/g,441.86±23.18μg/g vs 736.30±24.40μg/g,651.13±15.75μg/g,P<0.01;mRNA of Gremlin:64.86±2.83,55.82±5.39 vs 80.40±5.46,70.37±4.01,P<0.01;positive protein expression rate of Gremlin:20.78%±1.60%,17.43%±2.02%vs 38.46%±1.70%,29.50%±2.64%,P<0.01;relative protein expression of Gremlin:1.95±0.26,1.65±0.20 vs 2.81±0.24,2.22±0.63,P<0.01),and the mRNA and protein expression of BMP-7 was higher in the two DHC-treated groups(mRNA:73.52±4.56,81.78±5.38 vs 54.00±4.34,62.28±4.51,P<0.01;positive protein expression rate:41.44%±4.77%,47.49%±4.59%vs28.97%±3.14%,35.85%±3.50%,P<0.01;relative protein expression:0.71±0.06,0.81±0.07 vs 0.48±CONCLUSION:The therapeutic mechanism of DHC forhepatic fibrosis in rats may be associated with inhibitionof the expression of Gremlin and up-regulation of the expression of BMP-7.展开更多
The gene encoding bone morphogenetic protein-7(BMP7) is expressed in the developing kidney in embryos and also in the mature organ in adults. During kidney development, expression of BMP7 is essential to determine the...The gene encoding bone morphogenetic protein-7(BMP7) is expressed in the developing kidney in embryos and also in the mature organ in adults. During kidney development, expression of BMP7 is essential to determine the final number of nephrons in and proper size of the organ. The secreted BMP7 acts on the nephron progenitor cells to exert its dual functions: To maintain and expand the progenitor population and to provide them with competence to respond to differentiation cues, each relying on distinct signaling pathways. Intriguingly, in the adult organ, BMP7 has been implicated in protection against and regeneration from injury. Exogenous administration of recombinant BMP7 to animal models of kidney diseases has shown promising effects in counteracting inflammation, apoptosis and fibrosis evoked upon injury. Although the expression pattern of BMP7 has been well described, the mechanisms by which it is regulated have remained elusive and the processes by which the secretion sites of BMP7 impinge upon its functions in kidney development and diseases have not yet been assessed. Understanding the regulatory mechanisms will pave the way towards gaining better insight into the roles of BMP7, and to achieving desired control of the gene expression as a therapeutic strategy for kidney diseases.展开更多
AIM To present the incidence of heterotopic ossification after the use of recombinant human bone morphogenetic protein-7(rhB MP-7) for the treatment of nonunions.METHODS Bone morphogenetic proteins(BMPs) promote bone ...AIM To present the incidence of heterotopic ossification after the use of recombinant human bone morphogenetic protein-7(rhB MP-7) for the treatment of nonunions.METHODS Bone morphogenetic proteins(BMPs) promote bone formation by auto-induction. Recombinant human BMP-7 in combination with bone grafts was used in 84 patients for the treatment of long bone nonunions. All patients were evaluated radiographicaly for the development of heterotopic ossification during the standard assessment for the nonunion healing. In all patients(80.9%) with radiographic signs of heterotopic ossification, a CT scan was performed. Nonunion site palpation and ROM evaluation of the adjacent jointswere also carried out. Factors related to the patient(age, gender), the nonunion(location, size, chronicity, number of previous procedures, infection, surrounding tissues condition) and the surgical procedure(graft and fixation type, amount of rhB MP-7) were correlated with the development of heterotopic ossification and statistical analysis with Pearsons χ~2 test was performed.RESULTS Eighty point nine percent of the nonunions treated with rh BMP-7, healed with no need for further procedures. Heterotopic bone formation occurred in 15 of 84 patients(17.8%) and it was apparent in the routine radiologi-cal evaluation of the nonunion site, in a mean time of 5.5 mo after the rh BMP-7 application(range 3-12). The heterotopic ossification was located at the femur in 8 cases, at the tibia in 6, and at the humerus in οne patient. In 4 patients a palpable mass was present and only in one patient, with a para-articular knee nonunion treated with rhB MP-7, the size of heterotopic ossification affected the knee range of motion. All the patients with heterotopic ossification were male. Statistical analysis proved that patient's gender was the only important factor for the development of heterotopic ossification(P = 0.007). CONCLUSION Heterotopic ossification after the use of rh BMP-7 in nonunions was common but it did not compromise the final clinical outcome in most cases, and affected only male patients.展开更多
Objective: To express the recombinant human bone morphogenetic protein-7(rhBMP-7) in Chinese hamster ovary(CHO) cells, and to establish the in vitro biological activity assay of rhBMP-7.Methods: Human BMP-7 cDNA was s...Objective: To express the recombinant human bone morphogenetic protein-7(rhBMP-7) in Chinese hamster ovary(CHO) cells, and to establish the in vitro biological activity assay of rhBMP-7.Methods: Human BMP-7 cDNA was subcloned into p114 mammalian expression vector and transfected to CHO cells by using the Lipofectamine 2000 transfection method. CHO cell supernatants were harvested and analyzed to identify the molecule mass of secreted rhBMP-7 and examine its biological activity in vitro to stimulate the synthesis of alkaline phophatase(ALP), a characteristic of osteoblast phenotypes. Results: rhBMP-7 was produced stably in CHO cells, as a processed mature disulfide-linked homodimer, with an apparent molecular mass of 36 000. Examination of the rhBMP-7 biological activity showed that rhBMP-7 specifically stimulated the production of ALP(4-fold increase at 100 ng of rhBMP-7/ml). Conclusion: The rhBMP-7 from CHO expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates rhBMP-7 has the potential bone regeneration activity.展开更多
Objective To express the recombinant human bone morphogenetic protein-7 (rhBMP-7) in Chinese hamster ovary (CHO) cells and to establish the in vitro biological activity assay of rhBMP-7. Methods Human BMP-7 cDNA was s...Objective To express the recombinant human bone morphogenetic protein-7 (rhBMP-7) in Chinese hamster ovary (CHO) cells and to establish the in vitro biological activity assay of rhBMP-7. Methods Human BMP-7 cDNA was subcloned into pcDNA3.1 mammalian expression vector and transfected to CHO cells by using the lipofectin transfection method. BMP-7 expression cell culture supernatants were harvested and purified for target protein. To analyze the bioactivity of the secreted rhBMP-7, a novel in vitro assay was established by measuring its alkaline phosphatase (ALP) stimulating of osteoblast cell line, W-20-17. Results BMP-7 stably expressing cell clone was selected, which secreted mature disulfide-linked homodimer form of hBMP-7 and had an apparent molecular weight of 36kDa. rhBMP-7 with >95% purity was obtained using 3 step chromatography method. Bioactivity assay showed that the purified protein specifically stimulated W-20-17 cell producing ALP, with a 4-fold increase of ALP activity at 100ng/ml or more, and the EC50 of 15.6ng/ml. Conclusion Purified rhBMP-7 from this CHO expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates potential bone regeneration activity.展开更多
Bone morphogenetic protein-7 is widely accepted as an inducer for bone marrow stem cells differ-entiating into osteoblasts and chondrocytes. Whether bone marrow stromal cells differentiate into neuron-like cells remai...Bone morphogenetic protein-7 is widely accepted as an inducer for bone marrow stem cells differ-entiating into osteoblasts and chondrocytes. Whether bone marrow stromal cells differentiate into neuron-like cells remains unclear. The current study examined the presence of positive cells for in-termediate filament protein and microtubule associated protein-2 in the cytoplasm of bone marrow stromal cells induced by bone morphogenetic protein-7 under an inverted microscope, while no expression of glial fibrillary acidic protein was found. Reverse transcription PCR electrophoresis also revealed a positive target band for intermediate filament protein and microtubule-associated protein 2 mRNA. These results confirmed that bone morphogenetic protein-7 induces rat bone marrow stromal cells differentiating into neuron-like cells.展开更多
Objective:To clone the full-length human bone morphogenelic protein-7 (BMP-7) gene and analyse its sequence,to aid in investigation of its function and structure,Methods:Total RNA was isolated from Chinese fetal kidne...Objective:To clone the full-length human bone morphogenelic protein-7 (BMP-7) gene and analyse its sequence,to aid in investigation of its function and structure,Methods:Total RNA was isolated from Chinese fetal kidney by the acid guanidinium thiocyanate phenal-chloroform method.Two overlapping segments of human BMP-7 cDNA were obtained by reverse transcription (RT)-PCR.Following application,the two segments were ligated to each other and subcloned into PGEM-T easy vector to form PEGM-T easy/hBMP-7 recombinant plasmid.Sanger dideory chain-ter-mination method was used to sequence the cDNA.Results:There was 750bp fragment obtained RTPCR using #w primer from 5'' eng of BMP-7 gene(PCR by using # 2 and #1),and 540bp fragment from 3'' end was generated by RT-PCR using #4 primer(PCR using #3and #4).Full-length cDNA encoding BMP-7 was obained by religation of two segments,When compared with hBMP-7 sequence in Gene bank(XM30619),our full-length BMP-7 cDNA has a G instead of a T at nuclealide 862.This change results in valine substituting for phenylalanine in the protein.Conclusion:This is the first time that BMP-7 cDNA was successfully cloned from Chinese fetal kidney.BMP-7 cDNA plays an important role in healing injuries of the asteo-articular system.This makes BMP-7 is an attractive target for various clinical applications.展开更多
The extracellular matrix-associated bone morphogenetic proteins(BMPs) govern a plethora of biological processes. The BMPs are members of the transforming growth factor-β protein superfamily, and they actively partici...The extracellular matrix-associated bone morphogenetic proteins(BMPs) govern a plethora of biological processes. The BMPs are members of the transforming growth factor-β protein superfamily, and they actively participate to kidney development, digit and limb formation, angiogenesis, tissue fibrosis and tumor development. Since their discovery, they have attracted attention for their fascinating perspectives in the regenerative medicine and tissue engineering fields. BMPs have been employed in many preclinical and clinical studies exploring their chondrogenic or osteoinductive potential in several animal model defects and in human diseases. During years of research in particular two BMPs, BMP2 and BMP7 have gained the podium for their use in the treatment of various cartilage and bone defects. In particular they have been recently approved for employment in non-union fractures as adjunct therapies. On the other hand, thanks to their potentialities in biomedical applications, there is a growing interest in studying the biology of mesenchymal stem cell(MSC), the rules underneath their differentiation abilities, and to test their true abilities in tissue engineering. In fact, the specific differentiation of MSCs into targeted celltype lineages for transplantation is a primary goal of the regenerative medicine. This review provides an overview on the current knowledge of BMP roles and signaling in MSC biology and differentiation capacities. In particular the article focuses on the potential clinical use of BMPs and MSCs concomitantly, in cartilage and bone tissue repair.展开更多
AIM: To investigate the influence of bone morphogenetic protein type IA receptor [BMPR-IA(ALK3)] conditional knockout in lens on expression of bone morphogenetic protein 4(BMP4) in lens during the development of the v...AIM: To investigate the influence of bone morphogenetic protein type IA receptor [BMPR-IA(ALK3)] conditional knockout in lens on expression of bone morphogenetic protein 4(BMP4) in lens during the development of the vertebrate eye.METHODS: Cre-positive mice were mated with Crenegative mice to generate 50% Cre-positive(conditional knockout, CKO) 4 embryos, 8 eyes and 50% Cre-negative offspring(wild type, WT) 4 embryos, 8 eyes. The embryos were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 4 μm.Removal of paraffin wax and dehydrating for sections,and then the procedure of in situ hybridization was processed, BMP4 MK1784-m(BOSTER) was used, and observed the expression of BMP4 in the lens in experimental group and control group. We selected SPSS11.0 software for statistical analysis, P<0.05 showed that the difference was statistically significant.· RESULTS: Four embryos of each genotype were examined, totally we had 8 embryos, 16 eyes. We got the uniform outcomes in all the embryos. We found ALK3 was required during lens growing, but was not essential for the formation of lens. We observed that the expression of BMP4 in the lens was significantly reduced in all 8 ALK3 CKO lens, BMP4 expression was normal in all the 8 WT lens, P <0.01. This phenomenon became increasingly visible in accordance with embryo development. The most apparent alteration was present at stage E15.5.CONCLUSION: ALK3 is essential for lens growth. The influence of ALK3 on the expression of BMP4 is present during the development of mice lens.展开更多
Bone morphogenetic proteins(BMPs)are a family of potent,multifunctional growth factors belonging to transforming growth factor-(TGF-).They are highly conservative in structures.Over 20 members of BMPs with varying fun...Bone morphogenetic proteins(BMPs)are a family of potent,multifunctional growth factors belonging to transforming growth factor-(TGF-).They are highly conservative in structures.Over 20 members of BMPs with varying functions such as embryogenesis,skeletal formation,hematopoiesis and neurogenesis have been identified in human body.BMPs are unique growth factors that can induce the formation of bone tissue individually.BMPs can induce the differentiation of bone marrow mesenchymal stem cells into osteoblastic lineage and promote the proliferation of osteoblasts and chondrocytes.BMPs stimulate the target cells by specific membrane-bound receptors and signal transduced through mothers against decapentaplegic(Smads)and mitogen activated protein kinase(MAPK)pathways.It has been demonstrated that BMP-2,BMP-4,BMP-6,BMP-7,and BMP-9 play an important role in bone formation.This article focuses on the molecular characterization of BMPs family members,mechanism of osteogenesis promotion,related signal pathways of osteogenic function,relationships between structure and osteogenetic activity,and the interactions among family members at bone formation.展开更多
基金supported by grants from the National Key Research and Development Program of China(2021YFA1100603)the Nature Science Foundation of China(82071092)+2 种基金the Fundamental Research Funds for the Central Universities(YJ201878)Key Project of Sichuan province(2019YFS0311,2019YFS0515)Technology Innovation Research and Development Project of Chengdu(2019-YF05-00705-SN)。
文摘Pulp loss is accompanied by the functional impairment of defense,sensory,and nutrition supply.The approach based on endogenous stem cells is a potential strategy for pulp regeneration.However,endogenous stem cell sources,exogenous regenerative signals,and neovascularization are major difficulties for pulp regeneration based on endogenous stem cells.Therefore,the purpose of our research is to seek an effective cytokines delivery strategy and bioactive materials to reestablish an ideal regenerative microenvironment for pulp regeneration.In in vitro study,we investigated the effects of Wnt3a,transforming growth factor-beta 1,and bone morphogenetic protein 7(BMP7)on human dental pulp stem cells(h-DPSCs)and human umbilical vein endothelial cells.2D and 3D culture systems based on collagen gel,matrigel,and gelatin methacryloyl were fabricated to evaluate the morphology and viability of h-DPSCs.In in vivo study,an ectopic nude mouse model and an in situ beagle dog model were established to investigate the possibility of pulp regeneration by implanting collagen gel loading BMP7.We concluded that BMP7promoted the migration and odontogenic differentiation of h-DPSCs and vessel formation.Collagen gel maintained the cell adhesion,cell spreading,and cell viability of h-DPSCs in 2D or 3D culture.The transplantation of collagen gel loading BMP7 induced vascularized pulp-like tissue regeneration in vivo.The injectable approach based on collagen gel loading BMP7 might exert promising therapeutic application in endogenous pulp regeneration.
基金Supported by National Natural Science Foundation of China(30500414)Scientific Research Project in Department of Education of Liaoning Province(05L508,20061010)
文摘Objective To evaluate the effects of retinoic acid(RA)on expression of bone morphogenetic protein 7(BMP-7)in rat fetus with cleft palate,and the effects of RA on proliferation and apoptosis of osteoblasts.Methods All-trans RA(ATRA)was used to induce congenital cleft palate in Wistar rat.BMP-7 mRNA expression in maxillary bone tissue of fetal rats was measured by Northern blotting analysis.Flow cytometry and MTT assay were used to measure the apoptosis and proliferation of ATRA-treated MC-3T3-E1 cells.BMP-7 mRNA and protein expressions in ATRA-treated MC-3T3-E1 cells were detected by RT-PCR and Western blotting analysis.Results ATRA could induce cleft palate of rat fetus.The incidence rate of cleft palate induced by 100 mg/kg ATRA(45.5%)was significantly higher than 50 mg/kg ATRA(12.5%,P<0.05).BMP-7 mRNA expression decreased in maxillary bone tissue of rat fetus with cleft palate.MC-3T3-E1 cells proliferation treated with 1×10-6 mol/LATRA decreased by 60%,the cell apoptosis increased by 2 times.BMP-7 mRNA and protein levels in MC-3T3-E1 cells treated with 1×10-6 mol/L ATRA decreased by 60% and 80%,respectively,compared with ATRA-untreated cells(P<0.05).Conclusions BMP-7 may play an important role in embryonic palate development.RA may possess the ability to down-regulate cell proliferation through regulation of BMP-7 gene expression.
基金a grant from National Natural Sciences Foundation of China (No. 30572065/ C03031103)
文摘To construct the recombinant adeno-associated virus (rAAV) vector with human bone morphogenetic protein 7 (BMP7) and observe the BMP7 mRNA expression in vitro, BMP7 CDS se-quence was cloned into expression plasmid pAAV-MCS of AAV Helper Free System. The recombi-nant plasmid was identified with enzyme digestion and sequencing. The recombinant plasmid, pAAV-RC, pHelper were co-transfected into AAV-293 cells according to the calcium phosphate-based protocol. The viral stock was collected by 4 rounds of freeze/thaw. After purified and concentrated, the recombinant virus titer was determined by dot-blot assay. HEK293 cells were transfected with the recombinant virus at different MOI, and the expression of BMP7 mRNA was detected by RT-PCR. The results showed rAAV-BMP7 was constructed and packaged successfully. The physical particle titer was 2.5×1011 vector genomes/mL. There was different expression level of BMP7 mRNA after transfecton. These data suggested that recombinant AAV mediated a stable expression of hBMP7 mRNA in 293 cells. The AAV production method may pave the way of an effective strategy for the jaw bone defection around dental implants.
文摘BACKGROUND:Some researches demonstrate that exogenous bone morphogenetic protein 7(BMP-7) can protect ischemic cerebral nerve tissue and promote recovery of motor energy function;however,there is lack of direct evidences of endogenous BMP-7 effect. OBJECTIVE:To observe the expression of endogenous BMP-7 in nerve tissue with ischemic-hypoxic injury and investigate the possible effects on damaged nerve tissue. DESIGN:Observational contrast animal study. SETTING:Department of Anatomy and Histoembryology,Peking University Health Science Center. MATERIALS:The experiment was carried out in the Nerve Researching Laboratory of Anatomy Department,Peking University Health Science Center from October 2006 to March 2007. A total of 25 adult male SD rats weighing 250-300 g and several newborn SD rats were selected from Experimental Animal Center,Peking University Health Science Center. Rabbit-anti-BMP-7 polyclonal antibody was provided by Wuhan Boster Company. METHODS:① Adult rats were randomly divided into ischemia group(n =10),sham operation group(n = 10) and normal group(n =5). Right external-internal carotid artery occlusion was used to infarct middle cerebral artery of adult rats in the ischemia group so as to copy focal cerebral infarction models. Line cork was inserted in crotch of internal and external carotid artery of adult rats in the sham operation group,while adult rats in the normal group were not given any treatments. ② Cerebral cortex of newborn rats was separated to obtain cell suspension. Cells which were cultured for 10 days were divided into control group and hypoxia/reoxygenation group. And then,cells in the hypoxia/reoxygenation group were cultured in hypoxic incubator for 4 hours and given reoxygenation for 24 hours. MAIN OUTCOME MEASURES:Immunohistochemical method was used to measure expression of BMP-7 in cerebral cortex at 24 hours after ischemia/reperfusion culture and in primary hypoxic culture. RESULTS:① At 24 hours after cerebral ischemia,expression of BMP-7 in cerebral cortex on ischemic side was stronger than that on non-ischemic side in adult rats;meanwhile,numbers of cell expression were increased. However,expression of BMP-7 was not detected in bilateral cerebral cortex of adult rats in both control group and sham operation group. ② After hypoxia of cerebral cortex in primary culture,positive products of BMP-7 were observed in plasma of neuron,but expression of BMP-7 was not found in normal cerebral cortex. CONCLUSION:Endogenous BMP-7 has protective effects on nerve tissue induced by ischemic-hypoxic injury.
基金Indian Council of Medical Research,2020-0282/SCR/ADHOC-BMSDepartment of Science and Technology,India,DST/INSPIRE Fellowship:2021/IF210073.
文摘Mesenchymal stem cells(MSCs)originate from many sources,including the bone marrow and adipose tissue,and differentiate into various cell types,such as osteoblasts and adipocytes.Recent studies on MSCs have revealed that many transcription factors and signaling pathways control osteogenic development.Osteogenesis is the process by which new bones are formed;it also aids in bone remodeling.Wnt/β-catenin and bone morphogenetic protein(BMP)signaling pathways are involved in many cellular processes and considered to be essential for life.Wnt/β-catenin and BMPs are important for bone formation in mammalian development and various regulatory activities in the body.Recent studies have indicated that these two signaling pathways contribute to osteogenic differen-tiation.Active Wnt signaling pathway promotes osteogenesis by activating the downstream targets of the BMP signaling pathway.Here,we briefly review the molecular processes underlying the crosstalk between these two pathways and explain their participation in osteogenic differentiation,emphasizing the canonical pathways.This review also discusses the crosstalk mechanisms of Wnt/BMP signaling with Notch-and extracellular-regulated kinases in osteogenic differentiation and bone development.
文摘AIM: To investigate the antifibrotic effects of bone morphogenetic protein-7 (BMP-7) on Schistosoma japonicum (S. japonicum )-induced hepatic fibrosis in BALB/C mice. METHODS: Sixty BALB/C mice were randomly divided into three groups, including a control group (group A, n = 20), model group (group B, n = 20) and BMP-7 treated group (group C, n = 20). The mice in group B and group C were abdominally infected with S. japonicum cercariae to induce a schistosomal hepatic fibrosis model. The mice in group C were administered human recombinant BMP-7. Liver samples were extracted from mice sacrificed at 9 and 15 wk after modeling. Hepatic histopathological changes were assessed using Masson's staining. Transforming growth factor-beta 1 (TGF-β1), alpha-smooth muscle actin (α-SMA), phosphorylated Smad2/3 (pSmad2/3) and Smad7 protein levels and localization were measured by Western blotting and immunohistochemistry, respectively, and their mRNA expressions were detected by reverse transcriptionpolymerase chain reaction (RT-PCR). RESULTS: The schistosomal hepatic fibrosis mouse model was successfully established, as the livers of mice in group B and group C showed varying degrees of typical schistosomal hepatopathologic changes such as egg granuloma and collagen deposition. The degree of collagen deposition in group C was higher than that in group A (week 9: 22.95±6.66vs 2.02±0.76; week 15: 12.84±4.36 vs 1.74±0.80; P<0.05), but significantly lower than that in group B (week 9: 22.95±6.66 vs 34.43±6.96; week 15: 12.84±4.36 vs 18.90±5.07;P<0.05) at both time points. According to immunohistochemistry data, the expressions of α-SMA, TGF-β1 and pSmad2/3 protein in group C were higher than those in group A (α-SMA: week 9: 21.24±5.73 vs 0.33±0.20; week 15: 12.42±4.88 vs 0.34±0.27; TGF-β1: week 9: 37.00±13.74 vs 3.73±2.14; week 15: 16.71±9.80 vs 3.08±2.35; pSmad2/3: week 9: 12.92±4.81 vs 0.83±0.48; week 15: 7.87±4.09 vs 0.90±0.45; P<0.05), but significantly lower than those in group B (α-SMA: week 9: 21.24±5.73 vs 34.39±5.74; week 15: 12.42±4.88 vs 25.90±7.01; TGF-β1: week 9: 37.00±13.74 vs 55.66±14.88; week 15: 16.71±9.80 vs 37.10±12.51; pSmad2/3: week 9: 12.92±4.81 vs 19.41±6.87; week 15: 7.87±4.09vs 13.00±4.98;P<0.05) at both time points; the expression of Smad7 protein in group B was higher than that in group A and group C at week 9 (8.46±3.95 vs 1.00±0.40 and 8.46±3.95 vs 0.77±0.42; P<0.05), while there were no differences in Smad7 expression between the three groups at week 15 (1.09±0.38 vs 0.97±0.42 vs 0.89±0.39; P>0.05). Although minor discrepancies were observed, the results of RT-PCR and Western blotting were mainly consistentwith the immunohistochemical results. CONCLUSION: Exogenous BMP-7 significantly decreased the degree of hepatic fibrosis in both the acute and chronic stages of hepato-schistosomiasis, and the regulatory mechanism may involve the TGF-β/Smad signaling pathway.
基金Supported by the National Natural Science Foundation of China,No.81560104 and No.81860115
文摘BACKGROUND Liver fibrosis is a refractory disease whose persistence can eventually induce cirrhosis or even liver cancer.Early liver fibrosis is reversible by intervention.As a member of the transforming growth factor-beta(TGF-β)superfamily,bone morphogenetic protein 7(BMP7)has anti-liver fibrosis functions.However,little is known about BMP7 expression changes and its potential regulatory mechanism as well as the relationship between BMP7 and TGF-βduring liver fibrosis.In addition,the mechanism underlying the anti-liver fibrosis function of BMP7 needs to be further explored.AIM To investigate changes in the dynamic expression of BMP7 during liver fibrosis,interactions between BMP7 and TGF-β1,and possible mechanisms underlying the anti-liver fibrosis function of BMP7.METHODS Changes in BMP7 expression during liver fibrosis and the interaction between BMP7 and TGF-β1 in mice were observed.Exogenous BMP7 was used to treat mouse primary hepatic stellate cells(HSCs)to observe its effect on activation,migration,and proliferation of HSCs and explore the possible mechanism underlying the anti-liver fibrosis function of BMP7.Mice with liver fibrosis received exogenous BMP7 intervention to observe improvement of liver fibrosis by using Masson’s trichrome staining and detecting the expression of the HSC activation indicator alpha-smooth muscle actin(α-SMA)and the collagen formation associated protein type I collagen(Col I).Changes in the dynamic expression of BMP7 during liver fibrosis in the human body were further observed.RESULTS In the process of liver fibrosis induced by carbon tetrachloride(CCl4)in mice,BMP7 protein expression first increased,followed by a decrease;there was a similar trend in the human body.This process was accompanied by a sustained increase in TGF-β1 protein expression.In vitro experiment results showed that TGF-β1 inhibited BMP7 expression in a time-and dose-dependent manner.In contrast,high doses of exogenous BMP7 inhibited TGF-β1-induced activation,migration,and proliferation of HSCs;this inhibitory effect was associated with upregulation of pSmad1/5/8 and downregulation of phosphorylation of Smad3 and p38 by BMP7.In vivo experiment results showed that exogenous BMP7 improved liver fibrosis in mice.CONCLUSION During liver fibrosis,BMP7 protein expression first increases and then decreases.This changing trend is associated with inhibition of BMP7 expression by sustained upregulation of TGF-β1 in a time-and dose-dependent manner.Exogenous BMP7 could selectively regulate TGF-β/Smad pathway-associated factors to inhibit activation,migration,and proliferation of HSCs and exert antiliver fibrosis functions.Exogenous BMP7 has the potential to be used as an antiliver fibrosis drug.
基金Supported by The Science and Technology Project of Guizhou Province,China,No.SZ[2008]3049
文摘AIM:To observe the effect of Danshao Huaxian capsule(DHC)on the expression of Gremlin and bone morphogenetic protein-7(BMP-7)in the liver of hepatic fibrosis rats.METHODS:A total of 75 male Wistar rats were randomly divided into a normal control group(A),a CCl4-induced hepatic fibrosis model group(B),a natural recovery group(C),a low-dose DHC-treated group(D),and a high-dose DHC-treated group(E),with 15 rats in each group.Liver fibrosis was induced by subcutaneous injections of carbon tetrachloride(CCl4)and a highlipid/low-protein diet for 8 wk,except for the rats in group A.Then,the rats in the two DHC-treated groups were administered 0.5 and 1.0 g/kg DHC by gastrogavage once per day for 8 successive weeks,respectively.By the end of the experiment,the level of transforming growth factorβ1(TGF-β1)in the liver homogenate was determined by an enzyme-linked immunosorbent assay.The mRNA and protein expression of Gremlin and BMP-7 in the liver tissue was determined by reversetranscription polymerase chain reaction,an immunohistochemical assay,and Western blot analysis.RESULTS:Compared with group A,the level of TGF-β1and the mRNA and protein expression of Gremlin were significantly higher in group B(TGF-β1:736.30±24.40μg/g vs 284.20±18.32μg/g,P<0.01;mRNA of Gremlin:80.40±5.46 vs 49.83±4.20,P<0.01;positive protein expression rate of Gremlin:38.46%±1.70%vs 3.83%±0.88%,P<0.01;relative protein expression of Gremlin:2.81±0.24 vs 0.24±0.06,P<0.01),and the mRNA and protein expression of BMP-7was significantly lower in group B(mRNA:54.00±4.34vs 93.99±7.03,P<0.01;positive protein expression rate:28.97%±3.14%vs 58.29%±6.02,P<0.01;relative protein expression:0.48±0.31 vs 1.05±0.12,P<0.01).Compared with groups B and C,the degree of hepatic fibrosis was significantly improved,and the level of TGF-β1 and the mRNA and protein expression of Gremlin were significantly lowered in the two DHCtreated groups(TGF-β1:523.14±21.29μg/g,441.86±23.18μg/g vs 736.30±24.40μg/g,651.13±15.75μg/g,P<0.01;mRNA of Gremlin:64.86±2.83,55.82±5.39 vs 80.40±5.46,70.37±4.01,P<0.01;positive protein expression rate of Gremlin:20.78%±1.60%,17.43%±2.02%vs 38.46%±1.70%,29.50%±2.64%,P<0.01;relative protein expression of Gremlin:1.95±0.26,1.65±0.20 vs 2.81±0.24,2.22±0.63,P<0.01),and the mRNA and protein expression of BMP-7 was higher in the two DHC-treated groups(mRNA:73.52±4.56,81.78±5.38 vs 54.00±4.34,62.28±4.51,P<0.01;positive protein expression rate:41.44%±4.77%,47.49%±4.59%vs28.97%±3.14%,35.85%±3.50%,P<0.01;relative protein expression:0.71±0.06,0.81±0.07 vs 0.48±CONCLUSION:The therapeutic mechanism of DHC forhepatic fibrosis in rats may be associated with inhibitionof the expression of Gremlin and up-regulation of the expression of BMP-7.
基金Supported by Grants-in-Aid for Young Scientists(B)(No.15K18454 to Tsujimura T)Scientific Research(B)(No.15H03001 to Hishikawa K)Scientific Research(C)(Nos.25461208 to Takase O,15K09244 to Yoshikawa M and 26462400 to Idei M)from the Japan Society for the Promotion of Science
文摘The gene encoding bone morphogenetic protein-7(BMP7) is expressed in the developing kidney in embryos and also in the mature organ in adults. During kidney development, expression of BMP7 is essential to determine the final number of nephrons in and proper size of the organ. The secreted BMP7 acts on the nephron progenitor cells to exert its dual functions: To maintain and expand the progenitor population and to provide them with competence to respond to differentiation cues, each relying on distinct signaling pathways. Intriguingly, in the adult organ, BMP7 has been implicated in protection against and regeneration from injury. Exogenous administration of recombinant BMP7 to animal models of kidney diseases has shown promising effects in counteracting inflammation, apoptosis and fibrosis evoked upon injury. Although the expression pattern of BMP7 has been well described, the mechanisms by which it is regulated have remained elusive and the processes by which the secretion sites of BMP7 impinge upon its functions in kidney development and diseases have not yet been assessed. Understanding the regulatory mechanisms will pave the way towards gaining better insight into the roles of BMP7, and to achieving desired control of the gene expression as a therapeutic strategy for kidney diseases.
基金The European Union(European Social Fund-ESF)Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework(NSRF)-Research Funding Program:Heracleitus Ⅱ
文摘AIM To present the incidence of heterotopic ossification after the use of recombinant human bone morphogenetic protein-7(rhB MP-7) for the treatment of nonunions.METHODS Bone morphogenetic proteins(BMPs) promote bone formation by auto-induction. Recombinant human BMP-7 in combination with bone grafts was used in 84 patients for the treatment of long bone nonunions. All patients were evaluated radiographicaly for the development of heterotopic ossification during the standard assessment for the nonunion healing. In all patients(80.9%) with radiographic signs of heterotopic ossification, a CT scan was performed. Nonunion site palpation and ROM evaluation of the adjacent jointswere also carried out. Factors related to the patient(age, gender), the nonunion(location, size, chronicity, number of previous procedures, infection, surrounding tissues condition) and the surgical procedure(graft and fixation type, amount of rhB MP-7) were correlated with the development of heterotopic ossification and statistical analysis with Pearsons χ~2 test was performed.RESULTS Eighty point nine percent of the nonunions treated with rh BMP-7, healed with no need for further procedures. Heterotopic bone formation occurred in 15 of 84 patients(17.8%) and it was apparent in the routine radiologi-cal evaluation of the nonunion site, in a mean time of 5.5 mo after the rh BMP-7 application(range 3-12). The heterotopic ossification was located at the femur in 8 cases, at the tibia in 6, and at the humerus in οne patient. In 4 patients a palpable mass was present and only in one patient, with a para-articular knee nonunion treated with rhB MP-7, the size of heterotopic ossification affected the knee range of motion. All the patients with heterotopic ossification were male. Statistical analysis proved that patient's gender was the only important factor for the development of heterotopic ossification(P = 0.007). CONCLUSION Heterotopic ossification after the use of rh BMP-7 in nonunions was common but it did not compromise the final clinical outcome in most cases, and affected only male patients.
文摘Objective: To express the recombinant human bone morphogenetic protein-7(rhBMP-7) in Chinese hamster ovary(CHO) cells, and to establish the in vitro biological activity assay of rhBMP-7.Methods: Human BMP-7 cDNA was subcloned into p114 mammalian expression vector and transfected to CHO cells by using the Lipofectamine 2000 transfection method. CHO cell supernatants were harvested and analyzed to identify the molecule mass of secreted rhBMP-7 and examine its biological activity in vitro to stimulate the synthesis of alkaline phophatase(ALP), a characteristic of osteoblast phenotypes. Results: rhBMP-7 was produced stably in CHO cells, as a processed mature disulfide-linked homodimer, with an apparent molecular mass of 36 000. Examination of the rhBMP-7 biological activity showed that rhBMP-7 specifically stimulated the production of ALP(4-fold increase at 100 ng of rhBMP-7/ml). Conclusion: The rhBMP-7 from CHO expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates rhBMP-7 has the potential bone regeneration activity.
文摘Objective To express the recombinant human bone morphogenetic protein-7 (rhBMP-7) in Chinese hamster ovary (CHO) cells and to establish the in vitro biological activity assay of rhBMP-7. Methods Human BMP-7 cDNA was subcloned into pcDNA3.1 mammalian expression vector and transfected to CHO cells by using the lipofectin transfection method. BMP-7 expression cell culture supernatants were harvested and purified for target protein. To analyze the bioactivity of the secreted rhBMP-7, a novel in vitro assay was established by measuring its alkaline phosphatase (ALP) stimulating of osteoblast cell line, W-20-17. Results BMP-7 stably expressing cell clone was selected, which secreted mature disulfide-linked homodimer form of hBMP-7 and had an apparent molecular weight of 36kDa. rhBMP-7 with >95% purity was obtained using 3 step chromatography method. Bioactivity assay showed that the purified protein specifically stimulated W-20-17 cell producing ALP, with a 4-fold increase of ALP activity at 100ng/ml or more, and the EC50 of 15.6ng/ml. Conclusion Purified rhBMP-7 from this CHO expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates potential bone regeneration activity.
基金Science and Technology Research and Development Program of Shihezi University, No. ZRKX2009YB23
文摘Bone morphogenetic protein-7 is widely accepted as an inducer for bone marrow stem cells differ-entiating into osteoblasts and chondrocytes. Whether bone marrow stromal cells differentiate into neuron-like cells remains unclear. The current study examined the presence of positive cells for in-termediate filament protein and microtubule associated protein-2 in the cytoplasm of bone marrow stromal cells induced by bone morphogenetic protein-7 under an inverted microscope, while no expression of glial fibrillary acidic protein was found. Reverse transcription PCR electrophoresis also revealed a positive target band for intermediate filament protein and microtubule-associated protein 2 mRNA. These results confirmed that bone morphogenetic protein-7 induces rat bone marrow stromal cells differentiating into neuron-like cells.
文摘Objective:To clone the full-length human bone morphogenelic protein-7 (BMP-7) gene and analyse its sequence,to aid in investigation of its function and structure,Methods:Total RNA was isolated from Chinese fetal kidney by the acid guanidinium thiocyanate phenal-chloroform method.Two overlapping segments of human BMP-7 cDNA were obtained by reverse transcription (RT)-PCR.Following application,the two segments were ligated to each other and subcloned into PGEM-T easy vector to form PEGM-T easy/hBMP-7 recombinant plasmid.Sanger dideory chain-ter-mination method was used to sequence the cDNA.Results:There was 750bp fragment obtained RTPCR using #w primer from 5'' eng of BMP-7 gene(PCR by using # 2 and #1),and 540bp fragment from 3'' end was generated by RT-PCR using #4 primer(PCR using #3and #4).Full-length cDNA encoding BMP-7 was obained by religation of two segments,When compared with hBMP-7 sequence in Gene bank(XM30619),our full-length BMP-7 cDNA has a G instead of a T at nuclealide 862.This change results in valine substituting for phenylalanine in the protein.Conclusion:This is the first time that BMP-7 cDNA was successfully cloned from Chinese fetal kidney.BMP-7 cDNA plays an important role in healing injuries of the asteo-articular system.This makes BMP-7 is an attractive target for various clinical applications.
文摘The extracellular matrix-associated bone morphogenetic proteins(BMPs) govern a plethora of biological processes. The BMPs are members of the transforming growth factor-β protein superfamily, and they actively participate to kidney development, digit and limb formation, angiogenesis, tissue fibrosis and tumor development. Since their discovery, they have attracted attention for their fascinating perspectives in the regenerative medicine and tissue engineering fields. BMPs have been employed in many preclinical and clinical studies exploring their chondrogenic or osteoinductive potential in several animal model defects and in human diseases. During years of research in particular two BMPs, BMP2 and BMP7 have gained the podium for their use in the treatment of various cartilage and bone defects. In particular they have been recently approved for employment in non-union fractures as adjunct therapies. On the other hand, thanks to their potentialities in biomedical applications, there is a growing interest in studying the biology of mesenchymal stem cell(MSC), the rules underneath their differentiation abilities, and to test their true abilities in tissue engineering. In fact, the specific differentiation of MSCs into targeted celltype lineages for transplantation is a primary goal of the regenerative medicine. This review provides an overview on the current knowledge of BMP roles and signaling in MSC biology and differentiation capacities. In particular the article focuses on the potential clinical use of BMPs and MSCs concomitantly, in cartilage and bone tissue repair.
基金Supported by National Natural Science Foundation of China(No.30872836)Natural Science Foundation of Liaoning Province,China(No.201102054)
文摘AIM: To investigate the influence of bone morphogenetic protein type IA receptor [BMPR-IA(ALK3)] conditional knockout in lens on expression of bone morphogenetic protein 4(BMP4) in lens during the development of the vertebrate eye.METHODS: Cre-positive mice were mated with Crenegative mice to generate 50% Cre-positive(conditional knockout, CKO) 4 embryos, 8 eyes and 50% Cre-negative offspring(wild type, WT) 4 embryos, 8 eyes. The embryos were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 4 μm.Removal of paraffin wax and dehydrating for sections,and then the procedure of in situ hybridization was processed, BMP4 MK1784-m(BOSTER) was used, and observed the expression of BMP4 in the lens in experimental group and control group. We selected SPSS11.0 software for statistical analysis, P<0.05 showed that the difference was statistically significant.· RESULTS: Four embryos of each genotype were examined, totally we had 8 embryos, 16 eyes. We got the uniform outcomes in all the embryos. We found ALK3 was required during lens growing, but was not essential for the formation of lens. We observed that the expression of BMP4 in the lens was significantly reduced in all 8 ALK3 CKO lens, BMP4 expression was normal in all the 8 WT lens, P <0.01. This phenomenon became increasingly visible in accordance with embryo development. The most apparent alteration was present at stage E15.5.CONCLUSION: ALK3 is essential for lens growth. The influence of ALK3 on the expression of BMP4 is present during the development of mice lens.
基金This work was supported by National Natural Science Foundation Funding(3110131631371805)Program for New Century Excellent Talents in University of Ministry of Education of China(NCET-11-0796)and Heilongjiang Province Postdoctoral Science Foundation.
文摘Bone morphogenetic proteins(BMPs)are a family of potent,multifunctional growth factors belonging to transforming growth factor-(TGF-).They are highly conservative in structures.Over 20 members of BMPs with varying functions such as embryogenesis,skeletal formation,hematopoiesis and neurogenesis have been identified in human body.BMPs are unique growth factors that can induce the formation of bone tissue individually.BMPs can induce the differentiation of bone marrow mesenchymal stem cells into osteoblastic lineage and promote the proliferation of osteoblasts and chondrocytes.BMPs stimulate the target cells by specific membrane-bound receptors and signal transduced through mothers against decapentaplegic(Smads)and mitogen activated protein kinase(MAPK)pathways.It has been demonstrated that BMP-2,BMP-4,BMP-6,BMP-7,and BMP-9 play an important role in bone formation.This article focuses on the molecular characterization of BMPs family members,mechanism of osteogenesis promotion,related signal pathways of osteogenic function,relationships between structure and osteogenetic activity,and the interactions among family members at bone formation.
