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1.
Nat Commun ; 10(1): 2829, 2019 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-31249296

RESUMO

Extracellular vesicles (EVs) are involved in the regulation of cell physiological activity and the reconstruction of extracellular environment. Matrix vesicles (MVs) are a type of EVs released by bone-related functional cells, and they participate in the regulation of cell mineralization. Here, we report bioinspired MVs embedded with black phosphorus (BP) and functionalized with cell-specific aptamer (denoted as Apt-bioinspired MVs) for stimulating biomineralization. The aptamer can direct bioinspired MVs to targeted cells, and the increasing concentration of inorganic phosphate originating from BP can facilitate cell biomineralization. The photothermal effect of the Apt-bioinspired MVs can also promote the biomineralization process by stimulating the upregulated expression of heat shock proteins and alkaline phosphatase. In addition, the Apt-bioinspired MVs display outstanding bone regeneration performance. Our strategy provides a method for designing bionic tools to study the mechanisms of biological processes and advance the development of medical engineering.


Assuntos
Vesículas Extracelulares/metabolismo , Fósforo/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Biomineralização , Osso e Ossos/química , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Vesículas Extracelulares/química , Feminino , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/química , Osteoblastos/metabolismo , Fosfatos/metabolismo , Fósforo/química , Ratos
2.
Mol Med Rep ; 19(6): 5440-5452, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059099

RESUMO

The aim of the present study was to investigate the effects of advanced glycation end products (AGEs) and berberine hydrochloride (BBR) on the osteogenic differentiation ability of human periodontal ligament stem cells (hPDLSCs) in vitro, and their underlying mechanisms. hPDLSCs were subjected to osteogenic induction and were treated with AGEs or AGEs + BBR. Following varying numbers of days in culture, alkaline phosphatase (ALP) activity assays, ALP staining, alizarin red staining, ELISAs, and reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blot analyses were performed to determine the osteogenic differentiation ability of hPDLSCs; RT­qPCR, western blot analysis, and immunofluorescence staining were conducted to investigate the underlying mechanisms. The canonical Wnt/ß­catenin pathway inhibitor XAV­939 and agonist CHIR­99021 were used to determine the contribution of the canonical Wnt/ß­catenin pathway to differentiation. Treatment with AGEs resulted in reduced ALP activity and Collagen I protein levels, decreased ALP staining, fewer mineralized nodules, and downregulated expression of osteogenic­specific genes [Runt­related transcription factor 2 (Runx2), Osterix, ALP, osteopontin (OPN), Collagen I and osteocalcin (OCN)] and proteins (Runx2, OPN, BSP and OCN); however, BBR partially rescued the AGE­induced decrease in the osteogenic potential of hPDLSCs. Furthermore, AGEs activated the canonical Wnt/ß­catenin signaling pathway and promoted the nuclear translocation of ß­catenin; BBR partially attenuated this effect. In addition, XAV­939 partially rescued the AGE­induced reduction in the osteogenic potential of hPDLSCs, whereas CHIR­99021 suppressed the BBR­induced increase in the osteogenic potential of hPDLSCs. The present study indicated that AGEs attenuated the osteogenic differentiation ability of hPDLSCs, in part by activating the canonical Wnt/ß­catenin pathway; however, BBR attenuated these effects by inhibiting the canonical Wnt/ß­catenin pathway. These findings suggest a role for BBR in periodontal regeneration induced by hPDLSCs in patients with diabetes mellitus.


Assuntos
Berberina/farmacologia , Osteogênese/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação para Baixo/efeitos dos fármacos , Produtos Finais de Glicação Avançada/farmacologia , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Queratina-19/metabolismo , Ligamento Periodontal/citologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Fator de Transcrição Sp7/genética , Fator de Transcrição Sp7/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
3.
Talanta ; 201: 397-405, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31122440

RESUMO

This article reports the identification, engineering and characterisation of recombinant single chain variable fragment (scFv) antibody against Zearalenone (ZEN), an oestrogenic mycotoxin, using phage display antibody technology. To increase the chance of obtaining clones that can bind to free toxin, the conjugated proteins of the target antigen, i.e. bovine serum albumin ZEN-BSA and ovalbumin ZEN-OVA, were switched during the biopanning. One phage-displayed scFv clone specific to free ZEN, designated yZEN2A8, could be isolated. The gene encoding the yZEN2A8 scFv was sub-cloned into the pET-21d (+) and pKP300 delta III vectors to generate the recombinant scFv and scFv-AP antibody formats, respectively. After ELISA optimisation by checkerboard titration, the sensitivities of the recombinant yZEN2A8 scFv antibody and scFv-AP fusion were improved approx. 2 and 60 folds, respectively. Competitive ELISA indicated that the median inhibition concentration (IC50) of recombinant yZEN2A8 scFv antibody and scFv-AP fusion after ELISA optimisation were 90 and 14 ng mL-1, with a limit of detection (LOD) of 20 and 2 ng mL-1, respectively. No cross-reactivity to other common mycotoxins was observed. Homology modelling illustrated specific binding of the recombinant antibody to ZEN and demonstrated the role of complementary determining regions (CDRs) of both the variable heavy and light chains in antibody-antigen interactions. Efficient application of scFv-AP for the detection of ZEN contamination in corns and wheat samples were investigated for the first time. The antibody in the form of scFv-AP can be used as a prototype for the development of a convenient reagent for the detection of ZEN contamination in various format, including biosensor-based.


Assuntos
Ração Animal/análise , Contaminação de Alimentos/análise , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Cadeia Única/imunologia , Zearalenona/análise , Fosfatase Alcalina/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Técnicas de Visualização da Superfície Celular/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/genética , Humanos , Simulação de Acoplamento Molecular , Biblioteca de Peptídeos , Ligação Proteica , Coelhos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Triticum/química , Zea mays/química , Zearalenona/imunologia , Zearalenona/metabolismo
4.
Mol Med Rep ; 19(6): 4637-4644, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30957189

RESUMO

Arbutin is a natural compound extracted from various plants, including bearberry leaves, that exerts multiple effects including skin whitening, anti­inflammatory and oxidative stress­protective properties. However, the effects of arbutin on osteoblasts remain unknown. The aim of the present study was to investigate the function and the mechanisms of arbutin on the proliferation and differentiation of MC3T3­E1 mouse osteoblast precursor cells in vitro. The proliferation of MC3T3­E1 cells treated with arbutin was assessed using a Cell Counting Kit­8 assay and a 5­ethynyl­2'­deoxyuridine labeling assay. Additionally, cell cycle and apoptosis were examined using flow cytometry analysis. The effects of arbutin on osteoblast differentiation were investigated using alkaline phosphatase (ALP) staining and by examining the mRNA expression levels of collagen type I α1 chain (COL1A1), bone γ­carboxyglutamate protein (BGLAP) and Sp7 transcription factor (SP7). To further investigate the molecular mechanism underlying arbutin function in promoting osteogenesis, the mRNA and protein expression levels of runt­related transcription factor 2 (RUNX2) and ß­catenin were analyzed by reverse transcription­quantitative polymerase chain reaction and western blotting. Arbutin significantly promoted MC3T3­E1 cell proliferation and increased the ratio of cells in S­phase. Treatment with arbutin increased ALP activity and the mRNA expression levels of COL1A1, BGLAP and SP7 in MC3T3­E1 cells. Furthermore, the protein and the mRNA expression levels of RUNX2 and ß­catenin increased significantly following treatment with arbutin. Collectively, the present findings suggested that arbutin was able to promote proliferation and differentiation of MC3T3­E1 cells via the Wnt/ß­catenin signaling pathway.


Assuntos
Arbutina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células 3T3 , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Fator de Transcrição Sp7/genética , Fator de Transcrição Sp7/metabolismo , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismo
5.
Fish Shellfish Immunol ; 89: 179-186, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30928666

RESUMO

Alkaline phosphatase (AP) is a major, recently recognized component of innate immunity. The intestinal AP (IAP) isoform plays a pivotal role in controlling gastrointestinal and systemic inflammation in terrestrial mammals. This is so essentially through detoxification (by dephosphorylation) of proinflammatory microbial components that can no longer be recognized by so-called toll-like receptors, thus preventing cellular inflammatory cascade activation. A unique feature of fish is the presence of AP in skin and epidermal mucus (skin mucus AP) but its actual functions and underlying mechanisms of action are presently unknown. Here, we gather and analyse knowledge available on skin mucus AP in order to provide a holistic view of this important protective enzyme. Our main conclusions are that skin mucus AP is responsive to biotic and abiotic factors, including nutrients and bioactive feed components, prebiotics and probiotics. Importantly, both skin mucus AP and IAP appear to correlate, thus raising the interesting possibility that skin mucus AP be used as a proxy for IAP in future nutritional studies. Blood serum AP also seems to correlate with skin mucus AP, though biological interpretation for such relationship is presently unknown. Finally, the precise isoform/s of AP present in skin should be identified and underlying molecular mechanisms of skin mucus AP actions deciphered.


Assuntos
Fosfatase Alcalina/genética , Fosfatase Alcalina/imunologia , Peixes/fisiologia , Imunidade Inata/genética , Muco/imunologia , Animais , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Peixes/imunologia , Estado Nutricional , Pele/imunologia
6.
Mol Cell Biochem ; 458(1-2): 143-157, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31004309

RESUMO

MicroRNAs (miRNAs) regulate osteogenic differentiation of bone cells, which has applications in orthodontics. Here we evaluated the miRNA expression profile of MC3T3-E1 osteoblasts under cyclic tensile stress with chip technology and found that miR-132-3p was up-regulated by 12% cyclic tensile stress. Alkaline phosphatase activity and osteocalcin expression in MC3T3-E1 cells were decreased under these conditions. Smad2 and Smad5 were identified as potential target genes of miR-132-3p. Native and phosphorylated Smad2 and Smad5 expression was negatively correlated with miR-132-3p levels in the cells under cyclic stretch; however, only Smad5 protein level was reduced upon miR-132-3p overexpression. The luciferase reporter assay confirmed a direct interaction between miR-132-3p and Smad5. Thus, miR-132-3p maybe regulates osteoblast differentiation via Smad5 in response to cyclic tensile stress.


Assuntos
Diferenciação Celular , MicroRNAs/metabolismo , Osteoblastos/metabolismo , Proteína Smad5/metabolismo , Estresse Mecânico , Resistência à Tração , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Linhagem Celular , Camundongos , MicroRNAs/genética , Osteoblastos/citologia , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad5/genética
7.
Appl Microbiol Biotechnol ; 103(8): 3341-3353, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30887174

RESUMO

Antigen-binding fragments (Fabs) are an important part of monoclonal antibody (mAb) therapeutics and can be cost-effectively produced using an Escherichia coli (E. coli) expression system. However, Fabs tend to form undesirable aggregates when expressed in the cytoplasm of E. coli, substantially reducing the yield of correctly folded proteins. To solve this problem, in this study, we used five Fab fragments targeting IGF1R, Her2, VEGF, RANKL, and PD-1 to develop a novel system employing the alkaline phosphatase (phoA) promoter and the heat-stable enterotoxin II (STII) leader sequence to facilitate the efficient expression and extracellular secretion of Fabs. Following phosphate starvation, all five Fab fragments were expressed in BL21(DE3), were largely secreted into the culture medium, and then, were further purified by affinity chromatography specific to the constant region of the light chain. The purified Fab products were evaluated and were found to have high purity, antigen-binding affinity, and in vitro bioactivity. The mechanism experiments revealed that (1) BL21(DE3) had significantly higher productivity than the K-12 strains investigated; (2) the secretion ability of the PhoA promoter was superior to that of the T7 promoter; and (3) signal peptide, STII, showed higher extracellular secretion efficiency than pelB. Our findings strongly suggested that the phoA-STII-facilitated extracellular production platform is highly promising for application in the manufacturing of Fab fragments for both academic and industrial purposes.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fab das Imunoglobulinas/metabolismo , Fosfatase Alcalina/genética , Afinidade de Anticorpos , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Meios de Cultura/química , Enterotoxinas/genética , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
8.
Arch Endocrinol Metab ; 63(1): 89-93, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30864637

RESUMO

Hypophosphatasia is a rare inborn error of metabolism characterized by low serum alkaline phosphatase activity due to loss-of-function mutations in the gene encoding the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). Extracellular accumulation of TNSALP substrates leads to dento-osseous and arthritic complications featuring tooth loss, rickets or osteomalacia, and calcific arthopathies. Mild hypophosphatasia usually has autosomal dominant inheritance, severe cases are either autosomal recessive or due to a dominant negative effect. Clinical manifestations of hypophosphatasia are extremely variable, ranging from life threatening to asymptomatic clinical presentations. The clinical presentation of the adult-onset hypophosphatasia is highly variable. Fractures, joint complications of chondrocalcinosis, calcifying polyarthritis and multiple pains may reveal minor forms of the disease in adults. It is important to recognize the disease to provide the best supportive treatment and to prevent the use of anti-resorption drugs in these patients. Bone-targeted enzyme-replacement therapy (asfotase alfa) was approved in 2015 to treat pediatric-onset hypophosphatasia. We present a case of a 41-year-old male diagnosed with adult form of hypophosphatasia with a rare ALPL mutation that has been previously described only once and review the literature on the adult form of the disease and its genetic mechanism.


Assuntos
Fosfatase Alcalina/genética , Hipofosfatasia/genética , Mutação/genética , Adulto , Humanos , Masculino , Linhagem
9.
Int J Mol Sci ; 20(6)2019 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-30917596

RESUMO

Cadmium is a common environmental pollutant that causes bone damage. However, the effects of cadmium on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) and its mechanism of action in this process are unclear. Here, we determined the effects of cadmium chloride (CdCl2) on the osteogenic differentiation of BMMSCs and the potential mechanism involved in this process. As determined in the present investigation, CdCl2, in a concentration-dependent manner, affected the viability of BMMSCs and their cytoskeletons. Exposure to 0.1 or 0.2 µM CdCl2 inhibited osteogenic differentiation of BMMSCs, which was reflected in the down-regulation of osteoblast-related genes (ALP, OCN, Runx2, OSX, and OPN); in suppression of the protein expression of alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2); and in decreased ALP activity and capacity for mineralization. Moreover, mRNA microarray was performed to determine the roles of these factors in BMMSCs treated with CdCl2 in comparison to control BMMSCs. As determined with the microarrays, the Wingless-type (Wnt), mothers against decapentaplegic and the C. elegans gene Sam (SMAD), and Janus kinase-Signal Transducers and Activators of Transcription (JAK-STAT) signaling pathways were involved in the effects caused by CdCl2. Moreover, during differentiation, the protein levels of Wnt3a, ß-catenin, lymphoid enhancer factor 1 (LEF1), and T-cell factor 1 (TCF1) were reduced by CdCl2. The current research shows that CdCl2 suppresses the osteogenesis of BMMSCs via inhibiting the Wnt/ß-catenin pathway. The results establish a previously unknown mechanism for bone injury induced by CdCl2.


Assuntos
Células da Medula Óssea/metabolismo , Cloreto de Cádmio/farmacologia , Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Via de Sinalização Wnt , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteocalcina/genética , Osteocalcina/metabolismo , Osteopontina/genética , Osteopontina/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
In Vivo ; 33(2): 349-352, 2019 Mar-Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30804111

RESUMO

BACKGROUND/AIM: A better understanding of cementogenesis and cementoblast differentiation would be useful for periodontal therapy. The aim of this study was to establish a cell culture system that reflects cementum formation in periodontal tissue and determine whether or not isolated and cultured primary human periodontal ligament (PDL) cells could be used for the study of the differentiation of cementoblast. MATERIALS AND METHODS: PDL cells were isolated from the outgrowths of tissue fragments of human PDL. PDL cells were incubated for up to 21 days in differentiation medium containing ß-glycerophosphate and ascorbic acid. The changes in the cells were detected by alkaline phosphatase (ALP) and von Kossa staining. Real-time polymerase chain reaction was also performed for cementum protein 1 (CEMP1), which is a specific marker of cementoblasts and their progenitors. RESULTS: On day 5, a small number of PDL cells, which were fibrous, were positive for ALP. On day 7, almost all cells were positive for ALP. On day 14, mineralization nodules appeared, as seen by positive von Kossa staining; the nodules increased in number and size by day 21. The expression of CEMP1 was detected on day 5, and its expression level increased gradually by day 7, reached a peak on day 14, and decreased by day 21. CONCLUSION: Human PDL cells were used to establish a culture system that reflects cementum formation. Our results suggested that this culture method is convenient and useful for the study of cementogenesis and cementoblast differentiation.


Assuntos
Diferenciação Celular/genética , Ligamento Periodontal/citologia , Cultura Primária de Células , Proteínas/genética , Fosfatase Alcalina/genética , Cementogênese/genética , Cemento Dentário/citologia , Cemento Dentário/metabolismo , Glicerofosfatos/genética , Humanos , Ligamento Periodontal/metabolismo , Células-Tronco/enzimologia
11.
BMC Musculoskelet Disord ; 20(1): 80, 2019 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-30764793

RESUMO

BACKGROUND: Hypophosphatasia (HPP) is a rare, systemic disease caused by mutation(s) within the ALPL gene encoding tissue-nonspecific alkaline phosphatase (ALP). HPP has a heterogeneous presentation, which coupled with its rarity, often leads to missed/delayed diagnosis and an incomplete understanding of its natural history. To better understand the epidemiology and clinical course of HPP, including timing of diagnosis after first reported manifestation, we present baseline data for patients enrolled in the Global HPP Registry. METHODS: Data were analyzed from patients with an HPP diagnosis confirmed by low serum ALP activity and/or an ALPL pathogenic variant, regardless of prior or current treatment, according to age at enrollment (children: < 18 y; adult: ≥18 y). All analyses were descriptive. RESULTS: Of 269 patients from 11 countries enrolled January 2015-September 2017, 121 (45.0%) were children and 148 (55.0%) were adults. The majority of children and adults were female (61.2 and 73.0%, respectively) and white (57.7 and 90.0%, respectively). Children had a median (min, max) age at earliest reported HPP manifestation of 7.2 months (- 2.3 mo, 16.0 y), which was > 12 months before diagnosis at age 20.4 months (- 0.2 mo, 16.0 y). In adults, the earliest reported manifestation occurred at a median (min, max) age of 37.6 years (0.2 y, 75.2 y), which preceded age at diagnosis (47.5 years [0.2 y, 75.2 y]) by ~ 10 years. Premature loss of deciduous teeth (48.2%, age ≥ 6 mo), bone deformity (32.5%), and failure to thrive (26.7%) were most commonly reported in the HPP-related disease history of children. Pain (74.5%), orthopedic procedures and therapies (44.6%), and recurrent and poorly healing fractures (36.5%) were most commonly reported in the HPP-related disease history of adults. CONCLUSIONS: The Global HPP Registry represents the largest observational study of patients with HPP, capturing real world data. This analysis shows that diagnostic delay is common, reflecting limited awareness of HPP, and that HPP is associated with systemic manifestations across all ages. Many patients diagnosed in adulthood had HPP manifestations in childhood, highlighting the importance of taking thorough medical histories to ensure timely diagnosis. TRIAL REGISTRATION: Clinicaltrials.gov : NCT02306720 , December 2014; ENCePP.eu: EUPAS13526 , May 2016 (retrospectively registered).


Assuntos
Diagnóstico Tardio , Hipofosfatasia/diagnóstico , Adolescente , Adulto , Fatores Etários , Idoso , Fosfatase Alcalina/genética , Criança , Pré-Escolar , Europa (Continente)/epidemiologia , Feminino , Predisposição Genética para Doença , Humanos , Hipofosfatasia/tratamento farmacológico , Hipofosfatasia/epidemiologia , Hipofosfatasia/genética , Lactente , Japão/epidemiologia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Mutação , América do Norte/epidemiologia , Fenótipo , Valor Preditivo dos Testes , Prognóstico , Estudos Prospectivos , Sistema de Registros , Fatores de Tempo , Adulto Jovem
12.
Arch Oral Biol ; 99: 126-133, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30682715

RESUMO

OBJECTIVE: To explore the effect of oxytocin (OT) on the proliferation, migration, and osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in vitro. DESIGN: PDLSCs were obtained by limiting dilution method. Immunofluorescence (IF), cell-counting kit-8 (CCK8), cell migration assay, Alizarin Red S staining, cetylpyridinium chloride (CPC) colorimetry, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot analysis were used to examine the effect of OT on oxytocin receptor (OTR) expression, cell proliferation, migration and osteogenic differentiation of PDLSCs. RESULTS: Our study showed that PDLSCs expressed OTR. One hundred nanomolar OT exhibited the maximal effect on migration, while only 50 nM OT significantly promoted proliferation of PDLSCs, as well as mineralized nodule formation and calcium deposition (p < 0.05). Furthermore, 50 nM OT significantly up-regulated expression of osteogenesis-related genes-alkaline phosphatase (ALP), Collagen I (Col I), runt related transcription factor 2 (Runx 2), osteopontin (OPN) and osteocalcin (OCN)-at specific time points compared with osteogenic inductive medium (p < 0.05). In addition, western blot analysis demonstrated that 50 nM OT enhanced protein levels of ALP, Col I, and Runx 2 at day 7 and day 14 (p < 0.01), as well as activating the phosphorylation of extracellular-signal-regulated kinase (ERK) and protein kinase B (AKT) pathway; notably, 50 nM OT inhibited phosphorylation of the phosphatidylinositol 3-kinase (PI3K) pathway (p < 0.05). CONCLUSIONS: OT promoted proliferation, migration, and osteogenic differentiation of PDLSCs in vitro. Furthermore, the effect of OT on osteogenic differentiation was mediated through ERK and AKT pathway. Thus, OT may have potential for use in periodontal regeneration.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ocitocina/farmacologia , Ligamento Periodontal/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Adolescente , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Células Cultivadas , Criança , Colágeno/genética , Colágeno/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Osteocalcina/genética , Osteocalcina/metabolismo , Osteopontina/genética , Osteopontina/metabolismo , Ligamento Periodontal/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Ocitocina/metabolismo , Transdução de Sinais , Células-Tronco/citologia
13.
Gene Ther ; 26(3-4): 75-85, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30538307

RESUMO

Gene silencing for plasmid-based vectors and the underlying mechanism are critical factors for development of effective gene therapy. The objective of this study is to explore the role of epigenetic regulation for transgene expression. Two reporter genes, mouse interleukin 10 and human secreted alkaline phosphatase under the control of human cytomegalovirus immediate early promoter for expression, were delivered to mouse liver by hrodydynamics-based procedure and reporter gene expression was monitored. Reporter gene expression reached its peak level one day after gene delivery and declined progressively thereafter, reaching the minimal level in about 3 weeks. Intra-peritoneal injection of vorinostat, valproic acid or sodium butyrate, the known histone deacetylase inhibitors, resulted in a dose-dependent reactivation of reporter gene expression. Repeated administration of histone deacetylase inhibitors blocked gene silencing and maintained reporter gene expression. Mechanistic studies reveal that reactivation of reporter genes is corelated with hyperacetylation of histones H3 and H4, and elevated binding of TATA-box binding protein to the promoter region. These results suggest that epigenetic regulation plays a critical role in controlling transgene expression in vivo and demonstrate that enzymes involved in epigenetic regulation such as histone deacetylase could serve as a target to achieve controlled transgene expression for gene therapy.


Assuntos
Inativação Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Fosfatase Alcalina/genética , Animais , Epigênese Genética/genética , Feminino , Regulação da Expressão Gênica/genética , Inativação Gênica/fisiologia , Genes Reporter/genética , Vetores Genéticos , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas , Humanos , Interleucina-10/genética , Camundongos , Regiões Promotoras Genéticas/genética , Transgenes/genética , Ácido Valproico
14.
J Endod ; 45(1): 73-78, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30558800

RESUMO

INTRODUCTION: An ideal root canal sealer creates a bacteria-resistant seal and exhibits antimicrobial activity, biocompatibility, and osteoconductivity. The aim of this study was to assess the effects of 3 root canal sealers on cell viability, inflammatory response, and osteogenic potential in MC3T3-E1 cells. METHODS: AH Plus (Dentsply Caulk, Milford, DE), MTA Fillapex (Angelus Solucxoes Odontologicas, Londrina, Brazil), and EndoSequence BC (Brasseler, Savannah, GA) were mixed according to the manufacturer's instructions, and samples were prepared as extraction media (final dilution: 1/10). Lipopolysaccharide (LPS) (100 ng/mL) treatment was used to induce an inflammatory response in this study. Cell viability was evaluated using the Water soluble tetrazolium-1 (WST-1) assay. The levels of inflammatory mediators (interleukin 6 and tumor necrosis factor alpha) and osteogenic marker genes (ALP and OCN) were measured by reverse-transcription polymerase chain reaction and real-time polymerase chain reaction. Osteogenic potential was evaluated by alkaline phosphatase staining and alizarin red staining. RESULTS: Calcium silicate-based sealers such as MTA Fillapex and EndoSequence BC showed strong cell viability compared with AH Plus. AH Plus, MTA Fillapex, and EndoSequence BC decreased the levels of LPS-induced inflammatory mediators (P < .05). The expression of osteogenic marker genes, alkaline phosphatase activity, and mineralized nodule formation decreased with LPS treatment. However, AH Plus and calcium silicate-based sealers increased the osteogenic potential reduced by LPS treatment (P < .05). CONCLUSIONS: Calcium silicate-based sealers exhibit anti-inflammatory effects and induce osteogenic differentiation in MC3T3-E1 cells.


Assuntos
Anti-Inflamatórios , Compostos de Cálcio/farmacologia , Diferenciação Celular/efeitos dos fármacos , Osteoblastos/fisiologia , Osteogênese/efeitos dos fármacos , Materiais Restauradores do Canal Radicular/farmacologia , Silicatos/farmacologia , Fosfatase Alcalina/genética , Animais , Diferenciação Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Expressão Gênica , Camundongos , Osteogênese/genética , Estimulação Química
15.
Mol Med Rep ; 19(2): 943-950, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30535499

RESUMO

The purpose of this study was to evaluate the effects of concentrated growth factor exudate (CGFe) on human periodontal ligament cells (hPDLCs) stimulated by tumor necrosis factor (TNF)­α. From the peripheral blood of healthy donors, CGFe was prepared according to the Sacco protocol after 7 days of incubation. The hPDLCs were cultured by a tissue explant method and identified with anti­vimentin and anti­cytokeratin antibodies. Cells were subjected to four different treatments: i) Control; ii) TNF­α (10 ng/ml); iii) CGFe (concentration 50%); and iv) CGFe+TNF­α. The proliferation of hPDLCs was measured with Cell Counting Kit­8 assays. Osteogenic differentiation and mineralization were determined by Alizarin Red S staining, alkaline phosphatase activity, western blotting and reverse transcription­quantitative polymerase chain reaction. CGFe enhanced cell proliferation and upregulated ALP activity, the mineralization level, and osteogenic­associated osteocalcin, runt­related transcription factor 2 and Osterix gene expression in hPDLCs under inflammatory conditions induced by TNF­α. The present study demonstrated that CGFe enhanced hPDLC proliferation and osteogenic differentiation in the presence of TNF­α­induced inflammation. As CGFe can be obtained from the venous blood of patients, it generates no immune reaction. Thus, it is more economical and beneficial to use CGFe in clinical periodontal regeneration practice than synthetic growth factors.


Assuntos
Fibroblastos/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Osteoblastos/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Adolescente , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Misturas Complexas/química , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/isolamento & purificação , Queratinas/genética , Queratinas/metabolismo , Leucócitos Mononucleares/química , Masculino , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Ligamento Periodontal/citologia , Ligamento Periodontal/metabolismo , Cultura Primária de Células , Fator de Transcrição Sp7/genética , Fator de Transcrição Sp7/metabolismo , Vimentina/genética , Vimentina/metabolismo
16.
Mol Med Rep ; 19(2): 951-958, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30569151

RESUMO

Insufficient bone volume remains a key issue when using dental implants. Adipose tissue­derived stem cells (ADSCs) can accelerate bone healing when combined with dental implants. To improve the application of ADSCs for dental uses, the present study aimed to identify optimal implantation conditions. Mesenchymal stem cell­derived exosomes can induce naïve stem cells to differentiate through the osteogenic lineage. In the present study, exosomes derived from 3T3L1 preadipocytes (3T3L1­exo) were purified and characterized. The effects and potential mechanisms of 3T3L1­exo on 3T3L1 cell ossification were examined by reverse transcription­quantitative polymerase chain reaction, western blotting, electron microscopy, RNA sequencing and histological analysis. The current study confirmed that 3T3L1­exo enhanced 3T3L1 preadipocyte osteogenic differentiation, as revealed by upregulation of osteogenic differentiation­associated genes and increased Alizarin Red staining. Furthermore, the microRNA (miR) expression profiles of 3T3L1­exo and 3T3L1 preadipocytes were sequenced and compared. The results of a further analysis demonstrated that miR­223 expression was reduced in 3T3L1 preadipocytes stimulated by 3T3L1­exo compared with in unstimulated cells. This finding suggested that 3T3L1­exo promoted 3T3L1 bone formation by decreasing miR­223 through a competitive mechanism, another miRNA, or another factor. The mechanism by which miR­223 is decreased warrants further investigation. In conclusion, the application of 3T3L1­exo may be useful for investigating preadipocyte­induced bone regeneration.


Assuntos
Fatores Biológicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Exossomos/química , Regulação da Expressão Gênica/efeitos dos fármacos , MicroRNAs/genética , Osteogênese/efeitos dos fármacos , Células 3T3-L1 , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Biomarcadores/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Camundongos , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/genética , Fator de Transcrição Sp7/genética , Fator de Transcrição Sp7/metabolismo
17.
Methods Mol Biol ; 1873: 69-92, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30341604

RESUMO

Chaperonopathies are diseases in which abnormal chaperones play an etiopathogenic role. A chaperone is mutated or otherwise abnormal (e.g., modified by an aberrant posttranslational modification) in structure/function. To understand the pathogenic mechanisms of chaperonopathies, it is necessary to elucidate the impact of the pathogenic mutation or posttranslational modification on the chaperone molecule's properties and functions. This impact is usually subtle because if it were more than subtle the overall effect on the cell and organism would be catastrophic, lethal. This is because most chaperones are essential for life and, if damaged in structure/function too strongly, there would be death of the cell/organism, and no phenotype, i.e., there would be no patients with chaperonopathies. Consequently, diagnostic procedures and analysis of defects of the abnormal chaperones require a multipronged method for assessing the chaperone molecule from various angles. Here, we present such a method that includes assessing the intrinsic properties and the chaperoning functions of chaperone molecules.


Assuntos
Proteínas Arqueais/química , Varredura Diferencial de Calorimetria/métodos , Microscopia de Força Atômica/métodos , Chaperonas Moleculares/química , Mutação , Processamento de Proteína Pós-Traducional , Fosfatase Alcalina/química , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Amiloide/química , Amiloide/genética , Amiloide/metabolismo , Animais , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Temperatura Alta , Humanos , Malato Desidrogenase/química , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Penaeidae/química , Estabilidade Proteica , Pyrococcus furiosus/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
18.
Theranostics ; 8(20): 5575-5592, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30555565

RESUMO

Lineage differentiation of bone marrow mesenchymal stem cells (BMMSCs) is the key to bone-fat reciprocity in bone marrow. To date, the regulators of BMMSC lineage switching have all been identified to be transcription factors, and researchers have not determined whether other genes control this process. This study aims to reveal a previously unknown role of tissue-nonspecific alkaline phosphatase (TNSALP) in controlling BMMSC lineage selection. Methods: We compared the characteristics of cultured BMMSCs from patients with hypophosphatasia (HPP), which is caused by mutations in the liver/bone/kidney alkaline phosphatase (ALPL) gene, and an ALPL knockout (ko) mouse model. We performed ALPL downregulation and overexpression experiments to investigate the regulatory role of ALPL in BMMSC lineage switching. Using the PathScan array, coimmunoprecipitation experiments and pathway-guided small molecule treatments, we explored the possible mechanism underlying the regulatory effects of ALPL on cell differentiation and evaluated its therapeutic effect on ALPL ko mice. Results: BMMSCs from both patients with HPP and ALPL ko mice exhibited defective lineage differentiation, including a decrease in osteogenic differentiation and a parallel increase in adipogenic differentiation. Mechanistically, TNSALP directly interacted with LRP6 and regulated the phosphorylation of GSK3ß, subsequently resulting in lineage switching of BMMSCs. Re-phosphorylation of GSK3ß induced by LiCl treatment restored differentiation of BMMSCs and attenuated skeletal deformities in Alpl +/- mice. Conclusion: Based on our findings, TNSALP acts as a signal regulator to control lineage switching of BMMSCs by regulating the LRP6/GSK3ß cascade.


Assuntos
Fosfatase Alcalina/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Hipofosfatasia/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Adolescente , Fosfatase Alcalina/genética , Animais , Criança , Modelos Animais de Doenças , Glicogênio Sintase Quinase 3 beta/genética , Humanos , Hipofosfatasia/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Masculino , Camundongos Knockout
19.
J Agric Food Chem ; 66(51): 13435-13443, 2018 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-30556692

RESUMO

The key step for the toxicity of Bacillus thuringiensis subsp. israelensis (Bti) is the interaction between toxins and putative receptors; thus, many studies focus on identification of new toxin receptors and engineering of toxins with higher affinity/specificity for receptors. In the larvae of Aedes aegypti, galectin-14 was one of the genes upregulated by Bti treatment. RNAi knockdown expression of galectin-14 and feeding recombinant galectin-14-thioredoxin fusion protein significantly affected survival of Ae. aegypti larvae treated with Bti toxins. Recombinant galectin-14 protein bound to brush border membrane vesicles (BBMVs) of Ae. aegypti larvae, ALP1 and APN2, and galectin-14 and Cry11Aa bound to BBMVs with a similarly high affinity. Competitive binding results showed that galectin-14 competed with Cry11Aa for binding to BBMVs and ALP1 to prevent effective binding of toxin to receptors. These novel findings demonstrated that midgut proteins other than receptors play an important role in modulating the toxicity of Cry toxins.


Assuntos
Aedes/metabolismo , Fosfatase Alcalina/metabolismo , Proteínas de Bactérias/metabolismo , Endotoxinas/metabolismo , Galectinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Insetos/metabolismo , Aedes/química , Aedes/efeitos dos fármacos , Aedes/genética , Fosfatase Alcalina/química , Fosfatase Alcalina/genética , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/toxicidade , Endotoxinas/química , Endotoxinas/toxicidade , Galectinas/química , Galectinas/genética , Proteínas Hemolisinas/química , Proteínas Hemolisinas/toxicidade , Proteínas de Insetos/química , Proteínas de Insetos/genética , Larva/química , Larva/genética , Larva/metabolismo , Ligação Proteica
20.
Cell Physiol Biochem ; 50(4): 1522-1534, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30359975

RESUMO

BACKGROUND/AIMS: This study aimed to investigate the effect of Nell-1 on the osteogenic behaviors of pre-osteoblasts on titanium (Ti) surfaces and to identify the underlying signaling pathway. METHODS: Nell-1 at different concentrations was added to culture medium to stimulate MC3T3-E1 subclone 14 on Ti surfaces. A CCK-8 colorimetric assay was used to detect cell proliferation. Alkaline phosphatase activity (ALP) assay and enzyme-linked immunosorbent assay (ELISA) were used to evaluate ALP activity and the osteocalcin (OCN) secretion, respectively. Indicators of osteoblastic differentiation were assessed using real-time polymerase chain reaction analysis (RT-PCR). Western blot (WB) assay was used to analyze the expression changes of the osteogenic proteins and the mitogen-activated protein kinase (MAPK) pathway. RESULTS: Nell-1 significantly increased the osteogenic gene and protein expression levels of ALP, OCN, Runx2, osteoprotegerin (OPG), collagen type I (Col-I), and Osterix (Osx) in pre-osteoblasts on Ti surfaces. The optimal concentration of Nell-1 was 100 ng/ ml. In addition, Nell-1 activated ERK and JNK, but not P38, in MC3T3-E1 cells on the Ti surface. Except for ALP and Col-I, the promotive effects of Nell-1 on the expression of osteogenic markers were suppressed by ERK inhibitor U0126. CONCLUSION: Certain concentrations of Nell-1 can promote the osteogenic differentiation of pre-osteoblasts on Ti surfaces by activating the MAPK/ERK signaling pathway.


Assuntos
Proteínas de Ligação ao Cálcio/farmacologia , Diferenciação Celular/efeitos dos fármacos , Glicoproteínas/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Titânio/química , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Butadienos/farmacologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Nitrilos/farmacologia , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Fator de Transcrição Sp7/genética , Fator de Transcrição Sp7/metabolismo , Propriedades de Superfície
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