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Purpose To evaluate the effect of ergosterol combined with risedronate on fracture healing. Methods Sixty male Sprague Dawley fracture model rats were assigned into group A (n=20), group B (n=20), and group C (n=20) at random. All rats were fed by gavage until their sacrifice as it follows: group A with ergosteroside and risedronate, group B with risedronate, and group C with saline solution. At weeks 2 and 4, 10 rats of each group were sacrificed. Healing effect and bone tissue changes in the fractures site were assessed by using hematoxylin and eosin stain histology. Enzyme-linked immunosorbent assay was used to detect the expression of serum bone morphogenetic protein-2 (BMP-2), bone morphogenetic protein-7 (BMP-7), and vascular endothelial growth factor (VEGF). Reverse transcriptase polymerase chain reaction was applied to detect the expression of osteoprotegerin (OPG) mRNA, osteocalcin (OCN) mRNA and core-binding factor subunit-?1 (CBF-?1) mRNA. Results In terms of serum BMP-2, BMP-7, and VEGF expression at weeks 2 and 4 after gavage, group A < group B < group C (P<0.05). At week 4 after gavage, serum VEGF expression in the three groups harbored positive relationship with serum BMP-2 and BMP-7 expression (P<0.05). Regarding serum OPG, OCN and CBF-?1 mRNA expression at weeks 2 and 4 after gavage, group A Subject(s)
Male
, Animals
, Rats
, Fracture Healing/drug effects
, Ergosterol/analysis
, Vascular Endothelial Growth Factor A
, Osteoprotegerin/isolation & purification
, Risedronic Acid/analysis
, Reverse Transcriptase Polymerase Chain Reaction
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BACKGROUND: Bone marrow mesenchymal stem cells have the potential to differentiate into neuron-like cells, which have been listed as the preferred stem cells for the treatment of spinal cord injury. However, due to their low differentiation efficiency, it is particularly important to find a factor with high induction ability. Based on literature review and our previous studies, it is speculated that bone morphogenetic protein 7 (BMP-7) gene may play a vital role in promoting the differentiation of bone marrow mesenchymal stem cells into neuron-like cells. OBJECTIVE: To investigate the differentiation of rat bone marrow mesenchymal stem cells into neurons induced by BMP-7 lentivirus vector transfection. METHODS: Bone marrow mesenchymal stem cells of Sprague-Dawley rats were cultured by whole bone marrow adherence method, and then were transfected with LV-GFP when multiplicities of infection were 50, 25, 10, and 1. Green fluorescent protein expression was observed using fluorescence inversion microscope in each group at 3 days after transfection, to confirm the best multiplicity of infection. Passage 3 bone marrow mesenchymal stem cells were divided into blank control group (routine culture), LV-GFP group, and LV-BMP-7-GFP group, followed by transfection at the best multiplicity of infection. After 24, 48, 72, 96, and 120 hours of transfection, MTT assay was used to detect cell survival rate in each group. Immunocytochemical assay was used to detect the expression of nerve cell markers (neurofilament protein 200, synaptophysin-1) after 3 days of transfection. RESULTS AND CONCLUSION: (1) After 3 days of LV-GFP transfection, GFP-positive cells were observed under fluorescence microscopy when multiplicities of infection were 10, 25, and 50, whereas no GFP-positive cells were found when the multiplicity of infection was 1. The average fluorescence intensity was the highest when the multiplicity of infection was 10 (P < 0.05), indicating that multiplicity of infection=10 had the best infection effect. (2) Immunocytochemical results showed that the expression of neurofilament-200 and synaptophysin-1 was negative in the blank control group and LV-GFP group, but positive in the LV-BMP-7-GFP group. The cell body and axon in the LV-BMP-7-GFP group were dyed bright brown. In summary, lentivirus-mediated BMP-7 transfection can promote the differentiation of rat bone marrow mesenchymal stem cells into neuron-like cells.
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BACKGROUND: Autologous bone transplantation combined with strong fixation is considered as the gold standard for the treatment of nonunion. At present, there are many cases in which bone nonunion is treated with bone morphogenetic proteins 2 and 7. OBJECTIVE: To describe the osteogenic pathway of bone morphogenetic proteins at the gene level, summarize the clinical cases of nonunion treated with bone morphogenetic protein, and compare the therapeutic effects of bone morphogenetic proteins 2 and 7 on nonunion, followed by evaluation and analysis. METHODS: The first author used a computer to search the full-text database of Chinese journals, WanFang database and PubMed database. The key words were “BMP, nonunion, pathway, external fixator, ORIF, bone graft, infected nonunion, bone defect, osteoblast, osteoporosis " and 59 articles were finally included in the result analysis. RESULTS AND CONCLUSION: Literature review indicates that the gene-level pathway of bone morphogenetic protein can provide therapeutic ideas in clinical practice. In the treatment of bone nonunion, bone morphogenetic proteins 2 and 7 are effective, but there are yet no specifications and standards for the use of bone morphogenetic proteins, such as usage amount and indications. From the overall treatment effect and the treatment effect on infected bone nonunion, we compare the use of bone morphogenetic proteins 2 and 7. The effect of bone morphogenetic protein 2 is better than that of bone morphogenetic protein 7, especially in the treatment of infected nonunion.
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Abstract Purpose: To investigate the protective effects of salvianolic acid A (SAA) on renal damage in rats with chronic renal failure (CRF). Methods: The five-sixth nephrectomy model of CRF was successfully established in group CRF (10 rats) and group CRF+SAA (10 rats). Ten rats were selected as sham-operated group (group S), in which only the capsules of both kidneys were removed. The rats in group CRF+SAA were intragastrically administrated with 10 mg/kg SAA for 8 weeks. The blood urine nitrogen (BUN), urine creatinine (Ucr), creatinine clearance rate (Ccr), and serum uperoxide dismutase (SOD) and malondialdehyde (MDA) were tested. The expressions of transforming growth factor-β1 (TGF-β1), bone morphogenetic protein 7 (BMP-7) and Smad6 protein in renal tissue were determined. Results: After treatment, compared with group CRF, in group CRF+SAA the BUN, Scr, serum MDA and kidney/body weight ratio were decreased, the Ccr and serum SOD were increased, the TGF-β1 protein expression level in renal tissue was decreased, and the BMP-7 and Smad6 protein levels were increased (all P < 0.05). Conclusion: SAA can alleviate the renal damage in CRF rats through anti-oxidant stress, down-regulation of TGF-β1 signaling pathway and up-regulation of BMP-7/Smad6 signaling pathway.
Subject(s)
Animals , Male , Rats , Caffeic Acids/therapeutic use , Smad6 Protein/metabolism , Transforming Growth Factor beta1/metabolism , Bone Morphogenetic Protein 7/metabolism , Kidney Failure, Chronic/drug therapy , Lactates/therapeutic use , Down-Regulation , Up-Regulation , Rats, Sprague-Dawley , Disease Models, Animal , Kidney Failure, Chronic/chemically induced , Kidney Failure, Chronic/metabolism , Kidney Function Tests , NephrectomyABSTRACT
OBJECTIVE: To observe the effect of herbal cake-partitioned moxibustion on renal function and expression of connective tissue growth factor (CTGF), integrin-linked kinase (ILK) and bone morphogenetic protein-7 (BMP-7) in chronic renal failure (CRF) rabbits, so as to reveal its mechanisms underlying improvement of CRF. METHODS: Twenty-four male New Zealand rabbits were randomly divided into control, model, medication and herbal cake-partitioned moxibustion (moxibustion) groups (n=6 rabbits in each group). The CRF model was established by gavage of suspension of Adenine (150 mg·kg-1·d-1) for 21 days. Herbal cake-partitioned moxibustion was applied to "Mingmen"(GV4) and bilateral "Shenshu"(BL23), "Pishu"(BL20) and for 5 moxa-cones every time. Rabbits of the medication group was treated by gavage of Losartan Potassium (2.33 mg·kg-1·d-1). All the treatments were conducted once daily,12 times a course for consecutive 3 courses with a two-day rest after each course of treatment. Serum creatinine (Scr), blood urea nitrogen (BUN) and 24 h urine protein contents were detected by using an automatic biochemical analyzer. The expression levels of CTGF, ILK and BMP-7 proteins and mRNA in the kidney tissue were determined by Western blot and quantitative real time-PCR, separately. RESULTS: Following modeling, Scr and BUN and 24 h urine protein contents were significantly increased in the model group in comparison with the control group (P0.05). CONCLUSION: Herbal cake-partitioned moxibustion can improve renal function in CRF rabbits, which may be related to its effects in suppressing the expression of ILK and CTGF, and in up-regulating the expression of BMP-7 in the kidney tissue.
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Objective: To investigate clinical efficacy of Duhuo Xuduan Tang in treatment of lumbar disc herniation with different syndrome types and its effect on serum pain factors, bone morphogenetic protein-7 (BMP-7) and Aggrecan. Method: A total of 121 patients with non-emergency lumbar disc herniation admitted to the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine from January 2017 to June 2018 were randomly divided into observation group (62 cases) and control group (59 cases). The two groups of patients were treated by repositioning maneuver before absolute bed rest. observation group was given Duhuo Xuduan Tang, 150 mL·time-1, 3 times·d-1 orally, while control group was orally given ibuprofen, 300 mg·time-1, vitamin B1, 10 mg·time-1, 2 times·d-1, drugs were taken for 5 days a week, and then stopped for 2 days. Both groups were treated continuously for 4 weeks. changes in visual analogue score (VAS), modified Oswestry dysfunction index (MODI), serum substance P (SP), dopamine (DA), serotonin (5-HT), BMP-7 and Aggrecan were observed before and after treatment with enzyme-linked immunosandwich assay (ELISA). Result: Compared with before treatment, VAS and modified Oswestry dysfunction index scores were lower in both groups (PPPPPConclusion: Duhuo Xuduan Tang has a certain efficacy on different types of lumbar disc herniation, with best efficacy for patients with liver and kidney yin deficiency.
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Objective To observe the bone marrow mesenchymal stem cells (BMSCs) modified by bone morphogenetic protein-7 (BMP-7) gene on the expression of renal BMP-7, transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF), further to explore its protective mechanism on renal injury in rats with chronic renal failure (CRF). Methods BMSCs with high expression of BMP-7 gene (BMSCs-BMP-7) and empty vector-BMSCs (BMSCs-EV) were obtained by lentiviral-mediated gene transfection. Thirty male Sprague-Dawley (SD) rats were randomly divided into 5 groups, 6 in each group: normal control (CON) group; PBS intervention (CRF with PBS infusion, CRF+PBS) group; BMSCs intervention (CRF with BMSCs infusion, CRF+BMSCs) group;BMSCs-EV intervention (CRF with BMSCs-empty vector infusion, CRF+BMSCs-EV) group and BMSCs-BMP-7 intervention (CRF with BMSCs-BMP-7 infusion, CRF+BMSCs-BMP-7) group. The CRF model was established by 5/6 nephrectomy. The CON group was a sham operation group. The corresponding 12-weeks interventions of each experimental group were performed after 2 weeks of modeling, the rats in the CON group and the CRF+PBS group were injected with 1 ml of PBS through the tail vein, and the other three groups were injected with 1 ml of the corresponding cell suspension once a week. At the time of sacrifice, blood and renal tissue samples were reserved. Serum creatinine (Scr) and blood urea nitrogen (BUN) were measured by routine biochemical methods, and the expression of BMP-7, VEGF, TGF-β1 in kidney was assayed by Western blotting. Results At the time of sacrifice, the levels of Scr and BUN in the CRF+PBS group were significantly higher than those in the CON group (all P<0.01); Compared with the CRF+PBS group, the Scr and BUN of the CRF+BMSCs group, CRF+BMSCs-EV group and CRF+BMSCs-BMP-7 group were decreased to different extents, the differences were statistically significant (all P<0.01);the Scr and BUN of the CRF+BMSCs-BMP-7 group were significantly lower than CRF+BMSCs group and CRF+BMSCs-EV group (all P<0.05). The expression of BMP-7 and VEGF were the lowest in the CRF+PBS group. Compared with the CRF+PBS group, the expression of BMP-7 and VEGF in the CRF+BMSCs group, CRF+BMSCs-EV group and CRF+BMSCs-BMP-7 group were significantly increased respectively (all P<0.05). The expression of the BMP-7 and VEGF in the CRF+BMSCs-BMP-7 group were higher than those in the CRF+BMSCs group and CRF+BMSCs-EV group (P<0.01). Compared with the CON group, the expression of TGF-β1 in the CRF+PBS group was significantly increased (P<0.01);compared with the CRF+PBS group, the expression of TGF-β1 in the CRF+BMSCs group, CRF+BMSCs-EV and CRF+BMSCs-BMP-7 group was significantly decreased (all P<0.01);the expression of TGF-β1 in the CRF+BMSCs-BMP-7 group was lower than the CRF+BMSCs and CRF+BMSCs-EV group (both P<0.01). Conclusions BMSCs modified by BMP - 7 has a protective effect on CRF rats; its protective mechanism may be related to antagonizing TGF-β1 and up-regulation of renal VEGF expression.
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Objective To analyze the effects of bone morphogenetic protein 7 (BMP 7) on insulin signaling pathway in mice and its involved molecular mechanisms. Methods To increase BMP7 expression in liver, adenovirus bearing BMP7 was injected into mice via tail vein. The impact of BMP7 overexpression on glucose metabolism was assayed by glucose tolerance test and insulin tolerance test. The levels of proteins involved in insulin signaling pathway and c-Jun N-terminal kinase ( JNK) signaling pathway were analyzed by Western blot. Results The blood glucose level was increased by BMP7 overexpression (P>0. 05), while the glucose tolerance and insulin tolerance were decreased by BMP7. The p-Akt and p-GSK3βin liver and epididymal white adipose tissue (WAT) were reduced in the BMP7-overexpressed mice (P>0. 01), indicating insulin signal transduction was inhibited. In gastrocnemius muscle, the insulin signal transduction was not altered by BMP7. Mechanistically, the JNK pathway was activated by BMP7 in liver and epididymal WAT (P>0. 01), while the JNK pathway in skeletal muscle was not changed. Conclusions In mice, BMP7 elevated blood sugar and decreased glucose and insulin tolerance. BMP7 inhibited the insulin signaling pathway in liver and WAT. These inhibitory effects on insulin signaling pathway was likely to be achieved by an activating JNK signaling pathway.
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Objective To investigate the clinical efficacy of alprostadil in the treatment of diabetic nephropathy,and its effect on serum bone morphogenetic protein 7(BMP-7)and transforming growth factor-β1(TGF-β1)levels in patients.Methods From January 2014 to January 2016,120 cases of diabetic nephropathy in the People's Hospital of Beilun District were selected in the study.According to different treatment methods,the patients were divided into study group and control group,with 60 cases in each group.The two groups were treated with basic treatment,the study group was treated with alprostadil for 8 weeks.The clinical efficacy and serum levels of BMP-7 and TGF-β1 were compared between the two groups.Results There were no significant differences in serum BMP-7 and TGF-β1 between the two groups before treatment(P>0.05).After treatment,the serum BMP-7[(17.54 ±3.90)pg/mL]in the study group was higher than that in the control group [(15.20 ±2.96)pg/mL](t=3.607,P<0.05),the level of TGF-β1[(7786.3 ±1951.2)pg/mL]was lower than that of the control group [(10021.5 ±2109.7)pg/mL](t=6.025,P<0.05).There were no significant differences in the levels of fasting blood glucose(FBG),total cholesterol(TC),triglyceride(TG)and glycosylated hemoglobin(HbA1c)between the two groups(all P >0.05).After treatment,the levels of UAER,Hcy,Scr,BUN in the study group were(89.62 ±17.74)g/min,(23.55 ±4.17)mol/L,(64.2 ±8.5)mol/L,(6.70 ±0.96)mmol/L,which were significantly lower than those in the control group [(118.50 ± 21.18)g/min,(29.69 ±4.82)mol/L,(74.7 ±9)mol/L,(7.52 ±0.89)mmol/L](t=8.097,7.462,6.57,4.852,all P<0.05).Conclusion Alprostadil has better clinical effect on diabetic nephropathy,and can significantly improve serum BMP-7 and TGF-β1 levels.
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Objective: To evaluate the effect of bone morphogenetic protein 7 (BMP-7)/poly (lactide-co-glycolide) (PLGA) microspheres on in vitro proliferation and chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs).
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OBJECTIVE To investigate the mechanism of nicotinamide mononucleotide (NMN) on inflammation and fibrosis between endogenous nicotinamide phosphoribosyltransferase (NAMPT) and bone morphogenetic protein 7 (BMP-7) in diabetic glomerular cells.METHODS ① In vivo,spontaneous diabetic C57/BL6 mice and wild C57/BL6 mice were divided into two groups.When blood glucose was above (34.2±1.9) mmol· L-1,renal histology of diabetic mice became obvious.The protein expressions of Nampt and nuclear transcription factors-kappa B p65 (NF-κB p65),silent mating type information regulation 2 homolog 1 (SIRT1) and BMP7 were analyzed by lengths of immunofluorescence.② In vitro,rats' glomerular cells HBZY-1 were incubated with glucose 200 mmol· L-1 for different lengths of time (0,24,48 and 72 h) and at different concentrations of NMN (0,50,100 and 200 iμmol· L-1).The protein levels of Nampt and BMP7 were detected by Western blotting and the protein expressions of NF-κB p65 and α-SMA were measured by immunofluorescence assay.The protein levels of Nampt,BMP7 and NF-κB p65 were detected by Western blotting after HBZY-1 cells were treated with NMN 100 μmol· L-1 and FK866 10 μmol· L-1 for 24 h.RESULTS ① In vivo,the glomeruli of diabetic C57/BL6 mice showed obvious atrophy.Fluorescence intensity of Nampt was increased (P＜0.05),but that of BMP7 and SIRT1 in renal glomeruli cells was decreased compared with the wild type (P＜0.01).② In vitro,HBZY-1 cells were cultured in glucose 200 mmol· L-1 for 48 and 72 h.The protein expression of NAMPT was increased,but that of BMP7 was decreased (P＜0.05,P＜0.01).Expressions of NF-κB p65 and α-SMA were increased (P＜0.01) by immunofluorescence.The expression of BMP7 was increased after treatment with glucose 200 mmol· L-1,followed by NMN 50,100 and 200 μmol · L-1 for 24 h (P＜0.01).The expressions of NAMPT and NF-κB p65 were decreased (P＜0.01).The expressions of Nampt and NF-κB p65 in glucose 5.6 mmol· L1 +FK866 and glucose 5.6 mmol· L-1+ NMN groups were increased (P＜0.01),but the expression of BMP7 did not change.CONCLUSION Upregulation of endogenous Nampt obviously intervenes in BMP7 expression in the process of glomerular inflammatory fibrosis in severe diabetes.NMN can affect the protein expression of BMP7 via a special Nampt signaling pathway.
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Objective:To investigate the effects of bone morphogenetic protein 7 (BMP-7) on the biological properties of osteoblasts exposed to high glucose.Methods:MC3T3 cells were cultured in the high glucose condition and were divided into 4 groups according to BMP-7 concentration (ng/ml):0 (control group),50,100 and 200 ng/mL BMP-7 (test groups).The cell proliferation and ALP activity were examind 1,3,5 and 7 d after exposure.The number of intracellular calcium nodules were observed with Alizarin red staining after osteogenesis induction for 21 days.The F-actin cytoskeleton of MC3T3 was stained with Rhodamine and examined 24 h after culture.The mRNA expression of OCN and Runx2 were quantified by RT-qPCR 48h after exposure to BMP-7.Results:BMP-7 significantly promoted proliferation of MC3T3 cells and enhanced ALP activity(P ＜ 0.05),increased the number and volume of intracellular calcium nodules.In the cells exposed to BMP-7,the osteoblast form was improved and F-actin cytoskeleton started to change with network structure.Compared with control group,the mRNA levels of OCN and Runx2 were up-regulated in BMP-7 groups (P ＜ 0.05).The mRNA levels of OCN and Runx2 were the highest in 200 ng/ml BMP-7 group(P ＜ 0.05).Conclusion:BMP 7 may inhibit the action of the high concentration of glucose on MC3T3 cells.BMP-7 may be benificial to osseointegration of dental implant in patietents with diabetics.
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Objective To investigate the impact of sphingosine kinase 1 (SPK1) modified adipose tissue-derived stromal cells(ADSC)on tissue engineered bone osteogenesis.Methods ADSC cells isolated from SD rat fat cells were divided into CON group and SPK1 group,and then the cells were respectively infected with 10 MOI CON and SPK1 lentivirus for 48 h.The infection efficiency was confirmed by using flow cytometry.Alizarin red and oil red O was used to stain the cells 14 days after ADSC infection,and osteogenic and adipogenic ability was evaluated by detecting A595nm and A490 nm.In the meantime,the activity change of alkaline phosphatase(ALP)was detected.The SD rat femoral defect model was created,and then after combining ADSC with β-TCP,the tissue engineered bone was pressed to the defect site.The repairment of bone defect was detected by X-ray in 4,6 weeks.After infection of CON and SPK1 virus,bone morphogenetic protein(BMP7)expression in ADSC of these two groups was detected.Results The infection efficiency of CON and SPK1 lentivirus was 94.4％ vs.94.9％ respectively by flow cytometry.The SPK1 protein expression level in CON group and SPK1 group was (0.73±0.10) vs.(1.29±0.17)(P＜0.05).The A value of CON and SPK1 group at 595 nm was (0.20±0.02) vs.(0.41±0.01) (P＜0.05),respectively.The A value of CON and SPK1 group at 490 nm was (0.72±0.01) vs.(0.51±0.02)(P＜0.05),respectively.The expression level of ALP in CON and SPK1 group was (1.42±-0.09) vs.(2.68±0.09) (P＜0.01),respectively.In the repairment of bone defect,high density tissue at rat bone defect was significantly larger in SPK1 group than in CON group in 4 weeks,and in 6 weeks,bone defect of SPK1 group was healed,but CON group still had defect.The expression level of BMP7 in CON and SPK1 group was (1.13±0.16) vs.(4.46±0.23)(P＜0.05),respectively,48 h after infection.Conclusion SPK1 modified ADSC has an enhanced osteogenesis ability in vitro and in vivo,which was related to the activation of BMP7.
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A 12-year-old castrated Toy Poodle was referred to the Kangwon National University Animal Hospital with an oligotrophic nonunion fracture in the distal 1/3 of the left radius and an intact ulna. After fixation by a locking plate and screws, adipose-derived mesenchymal stem-cell sheets expressing bone morphogenetic protein 7 (BMP-7) were transplanted to the fracture site to enhance the healing activity. The fracture was healed at 9 weeks after surgery. In the present case, the mesenchymal stem-cell sheets expressing BMP-7 promoted bone regeneration and healing in a nonunion fracture.
Subject(s)
Animals , Child , Dogs , Humans , Bone Morphogenetic Protein 7 , Bone Regeneration , Fractures, Ununited , Hospitals, Animal , Play and Playthings , Radius , UlnaABSTRACT
OBJECTIVE: The aim was to study the effects of application of ionizing radiation (gamma and electrons) as sterilizing agents at doses of 15 kGy, 25 kGy and 50 kGy, on lyophilized or frozen demineralized bone tissue for use in transplants. METHODS: Five human femoral diaphyses from different donors of musculoskeletal tissue were demineralized and preserved as lyophilized or frozen at -80 °C. The samples were divided into two groups: non-irradiated (control) and irradiated by means of gamma rays or an electron beam. The bone proteins were extracted and used to determine the concentrations of total protein and BMP 2 and 7. RESULTS: Decreases in total protein and BMP 2 and 7 concentrations were observed. The decreases in total protein concentrations, in comparison with the respective control groups, were significant in the lyophilized and frozen samples that were irradiated at a dose of 50 kGy of gamma radiation and electron beam, with reductions of more than 30%. Significant decreases in the levels of BMP 2 and 7 were also observed at higher doses and especially through use of the electron beam. CONCLUSION: The reductions in the concentrations of total proteins and osteoinductive proteins (BMP 2 and 7) were related to the radiation dose, i.e. they increased with higher doses of ionizing radiation type and the type of bone preservation. The largest reductions in concentrations were observed in the bones irradiated by means of an electron beam and at a dose of 50 kGy. However, this type of radiation and this high dose are not usual practices for sterilization of bone tissue.
OBJETIVO: Estudar os efeitos da aplicação das radiações ionizantes (gama e elétrons) como agentes esterilizantes, nas doses de 15 kGy, 25 kGy e 50 kGy, nos tecidos ósseos desmineralizados congelados e liofilizados para uso em transplantes. MÉTODOS: Cinco diáfises femorais humanas de doadores distintos de tecidos musculoesqueléticos foram desmineralizadas e preservadas como liofilizadas ou congeladas a -80 °C. As amostras foram divididas em grupos não irradiados (controle) e irradiados por raios gama ou feixe de elétrons. As proteínas ósseas foram extraídas e dosadas as concentrações de proteínas totais, BMP 2 e 7. RESULTADOS: Foi observada diminuição das concentrações de proteínas totais e BMP 2 e 7. A diminuição das concentrações de proteínas totais, quando comparada com o respectivo controle, foi significativa nos grupos de amostras liofilizadas e congeladas e irradiadas na dose de 50 kGy por radiação gama e feixe de elétrons com redução superiores a 30%. A diminuição significativa nas concentrações das BMP 2 e 7 também foi observada nas maiores doses e principalmente por feixe de elétrons. CONCLUSÃO: As reduções nas concentrações das proteínas totais e em proteínas osteoindutoras (BMP 2 e 7) foram relacionadas à dose de radiação, ou seja, aumentam com maiores doses, tipo de radiação ionizante e ao tipo de preservação dos ossos. As maiores reduções das concentrações foram observadas nos ossos irradiados por feixe de elétrons e na dose de 50 kGy. Porém esse tipo de radiação e essa alta dose não são práticas usuais para a esterilização dos tecidos ósseos.
Subject(s)
Humans , Male , Female , Bone and Bones , Radiation, Ionizing , Tissue BanksABSTRACT
Objective To investigate the effects of bone morphogenetic protein-7 ( BMP7 ) on insulin signaling transduction in C2C12 myotubes and HepG2 hepatocytes, and the underlying mechanisms were studied preliminarily.Methods The C2C12 myotubes and HepG2 cells were treated with BMP7 at different concentrations.The insulin signal transduction was analyzed by Western blot.Meanwhile, total RNA was extracted and quantitative PCR was employed for detecting the effects of BMP7 on gene expressions of effectors involved in insulin signal pathway.Furthermore, JNK signal pathway was also measured.Results The protein levels of p-IR, p-Akt and p-GSK3β, as well as glucose uptake, were significantly stimulated by insulin in the C2C12 myotubes and HepG2 cells.However, these stimulations induced by insulin were largely attenuated by BMP7.The mRNA levels of Akt1, Igf1r, Insr, and Irs1 were not altered by the treatment of BMP7.The JNK signal pathway was activated by a 5-min exposure of BMP7 in the HepG2 cells, and this activation was gradually reduced along with the treating time.Conclusion BMP7 attenuates the insulin signal transduction in the HepG2 cells and C2C12 myotubes, and this attenuation effect may be through JNK activation.
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AIM: To investigate the effects of bone morphogenetic protein 7 ( BMP-7 ) on the expression of transcription factor E2A and inhibitor of differentiation 2 (Id2) in the renal tubule epithelial cells(NRK-52E)exposed to high glucose, and to explore its possible mechanism of improving renal tubular fibrosis induced by high glucose.METH-ODS:The NRK-52E cells were divided into control group, high glucose (HG) group and high glucose with different doses of BMP-7 (10μg/L and 20μg/L) group.The cells in HG group and BMP-7 group were cultured for 12 h, 24 h and 48 h. The protein expression of Id2, E2A, E-cadherin,α-smooth muscle actin (α-SMA) and collagen-I was detected by Western blot.In addition, the mRNA expression of Id2 was detected by real-time PCR.RESULTS:Compared with control group, the mRNA and protein levels of Id2 and the protein level of E-cadherin were down-regulated, while the protein levels of E2A,α-SMA and collagen-I were up-regulated in HG group (P<0.05).Compared with HG group, the mRNA and pro-tein levels of Id2 and the protein level of E-cadherin were significantly up-regulated, while the protein expression of E2A,α-SMA and collagen-I was significantly down-regulated in 20 μg/L BMP-7 group ( P<0.05 ) .The correlation analysis showed that the Id2 protein level was negatively correlated with the E2A protein level (P<0.05).CONCLUSION:BMP-7 may intercept the process of renal tubule fibrosis induced by high glucose via promoting the expression of Id2 and inhibi-ting the expression of E2A at protein level.
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BACKGROUND:Liver fibrosis is a kind of chronic and active disease that is caused by various causes and characterized by the excessive accumulation of extracelular matrix. At present, use of Chinese herbs for the treatment of hepatic fibrosis has obvious advantages. Salvia miltiorrhiza has been shown to be highly effective in the treatment of hepatic fibrosis. However, the underlying mechanism needs further investigation. OBJECTIVE:To investigate the effects of TanshinoneⅡA on the expression levels of transforming growth factor-β/Smads signaling pathway related factors transforming growth factor-β, bone morphogenetic protein 7, Smad6 and Smad7 in the liver tissue of rats with hepatic fibrosis. METHODS:SD rats were randomly divided into three groups (n = 10): normal control, model and TanshinoneⅡA-treated groups. Rats in the model and TanshinoneⅡA-treated groups were subtaneously injected with olive oil-diluted 10% CCl4 ( 5 mL/kg) twice a week, 8 weeks in total, to build rat models of hepatic fibrosis. Four weeks after hepatic fibgrosis induction, rats in the TanshinoneⅡA-treated group received subtaneous injection of TanshinoneⅡA til eight weeks. Rats in the normal control group were subcutaneously injected with olive oil. RESULTS AND CONCLUSION: Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) detection showed that in the model group, the expression of transforming growth factor-β in the rat liver tissue was significantly increased (P < 0.01) and the expression of bone morphogenetic protein-7, Smad6 and Smad7 was significantly decreased (P < 0.01) compared with the normal control group. TanshinoneⅡA could obviously reverse the expression of those factors above-mentioned (P < 0.01). The results suggest that TanshinoneⅡA can be used for treatment of hepatic fibrosis by decreasing the expression of transforming growth factor-β and increasing the expression of bone morphogenetic protein-7, Smad6 and Smad7.
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Objective:To study the influence of bone morphogenetic protein-7 ( BMP-7 ) on chondro-cyte secretion and expression of type Ⅱ collagen ( Col-Ⅱ) , aggrecan ( AGG ) and SRY-related high mobility group-box gene 9 ( Sox9 ) mRNA in porous tantalum-chondrocyte composites.Methods: The articular chondrocytes were isolated from 3-week-old New Zealand immature rabbits and identified.The 2nd generation of chondrocytes with 1 ×106/mL inoculate concentration was seeded in porous tantalum and divided into 4 groups, and control group ( tantalum/chondrocyte) , 50μg/L BMP-7 group (50μg/L BMP-7/tantalum/chondrocyte) , 100 μg/L BMP-7 group ( 100 μg/L BMP-7/tantalum/chondrocyte ) , and 200 μg/L BMP-7 group ( 200 μg/L BMP-7/tantalum/chondrocyte ) .The proliferation of chondro-cytes was measured by CCK-8 assay.The chondrocyte growth and morphology were observed by scanning electron microscopy ( SEM) .The synthesis of glycosaminoglycan ( GAG) in chondrocytes was tested by dimethyl methylene blue ( DMMB) colorimetric quantification method.Col-Ⅱ, AGG and Sox9 mRNA in chondrocytes were detected by real-time PCR.Results: The chondrocytes were spindle-shaped in 24 hours of primary cell culture and most cells became polygonal shaped in 4 days.The chondrocytes were affirmed by alcian blue, safranin O and Col-Ⅱimmunocytochemistry staining.The result of CCK-8 assay showed that the level of cell proliferation in 100 μg/L BMP-7 groups were higher than those in the other groups ( P<0 .05 ) .The chondrocytes implanted into porous tantalum scaffolds with BMP-7 had better functions, by which cytoplasmic processes developed and extended to the surface and inner of porous tan-talum by SEM observation.DMMB quantitative determination of GAG showed that GAG amount of chon-drocytes in 100 μg/L BMP-7 groups was significantly higher than those in the other groups ( P<0 .05 ) . The expressions of Col-Ⅱ, AGG and Sox9 mRNA in chondrocytes were up-regulated in the experimental groups, compared with the control group and the best effect appeared when concentration of BMP-7 was 200μg/L.(P<0.05).Conclusion:BMP-7/tantalum/chondrocytes composites enhanced in vitro chon-drocyte proliferation and extracellular matrix greatly, and can promote chondrogenic gene expression.
ABSTRACT
AIM:To study the protective effects and mechanism of Fushen Jiangzhuo formula (FSJZ) on the rat renal interstitial fibroblasts with renal tubulointerstitial lesion .METHODS:The serum containing FSJZ and blank con-trol serum were prepared .The rat model of mesangial proliferative glomerulonephritis ( MsPGN) was established and ex-tended the time of modeling to 20th weeks for developing tubulointerstitial lesion naturally .The rat renal interstitial fibro-blasts at the end of 12, 16, 20 week of modeling were isolated and cultured .The effects of FSJZ on the expression of hepa-tocyte growth factor (HGF) and bone morphogenetic protein-7 (BMP-7) at mRNA and protein levels in the pathological re-nal interstitial fibroblasts were determined .RESULTS:The anti-fibrosis factors HGF and BMP-7 were changed in patho-logical renal interstitial fibroblasts .Renal tubulointerstitial lesion significantly down-regulated the expression of HGF and BMP-7, while the drug containing serum of FSJZ significantly up-regulated the expression of HGF and BMP-7.CONCLU-SION:Drug containing serum of FSJZ has protective effect on pathological renal interstitial fibroblasts by regulating the mRNA and protein expression of HGF and BMP-7.