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1.
Cell Physiol Biochem ; 53(4): 623-637, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31550089

RESUMO

BACKGROUND/AIMS: In articular cartilage, chondrocytes are the predominant cell type. A long-term stay in space can lead to bone loss and cartilage breakdown. Due to the poor regenerative capacity of cartilage, this may impair the crewmembers' mobility and influence mission activities. Beside microgravity other factors such as cosmic radiation and vibration might be important for cartilage degeneration. Vibration at different frequencies showed various effects on cartilage in vivo, but knowledge about its impact on chondrocytes in vitro is sparse. METHODS: Human chondrocytes were exposed to a vibration device, simulating the vibration profile occurring during parabolic flights, for 24 h (VIB) and compared to static controls. Phase-contrast microscopy, immunofluorescence, F-actin and TUNEL staining as well as quantitative real-time PCR were performed to examine effects on morphology, cell viability and shape as well as gene expression. The results were compared to earlier studies using semantic analyses. RESULTS: No morphological changes or cytoskeletal alterations were observed in VIB and no apoptotic cells were found. A reorganization and increase in fibronectin were detected in VIB samples by immunofluorescence technique. PXN, VCL, ANXA1, ANXA2, BAX, and BCL2 revealed differential regulations. CONCLUSION: Long-term VIB did not damage human chondrocytes in vitro. The reduction of ANXA2, and up-regulation of ANXA1, PXN and VCL mRNAs suggest that long-term vibration might even positively influence cultured chondrocytes.


Assuntos
Condrócitos/metabolismo , Vibração , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Anexina A2/genética , Anexina A2/metabolismo , Linhagem Celular , Condrócitos/citologia , Condrócitos/patologia , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Redes Reguladoras de Genes , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Vimentina/genética , Vimentina/metabolismo
2.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(7): 694-696, 2019 Jul 10.
Artigo em Chinês | MEDLINE | ID: mdl-31302913

RESUMO

OBJECTIVE: To explore the molecular basis for a pedigree affected with spondyloepiphyseal dysplasia congenita (SEDC). METHODS: The proband was subjected to whole exome sequencing. Suspected variant was verified by Sanger sequencing. RESULTS: All patients from the pedigree were found to carry a novel missense variant c.1394G>C (p.Gly465Ala) of the COL2A1 gene. The variant was not reported previously. Provean, Polyphen-2 and Mutation Taster software predicted that the variant is highly likely to be pathogenic. CONCLUSION: The c.1394G>C (p.Gly465Ala) variant of the COL2A1 gene probably underlies the SEDC in this pedigree.


Assuntos
Colágeno Tipo II/genética , Osteocondrodisplasias/congênito , Grupo com Ancestrais do Continente Asiático , Humanos , Osteocondrodisplasias/genética , Linhagem
3.
Int J Mol Sci ; 20(12)2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-31200471

RESUMO

Lactoferrin (LF) is known to modulate the bone anabolic effect. Previously, we and others reported that the effects of LF on the bone may be conferred by the stimulation of transforming growth factor ß (TGF-ß) signaling in the preosteoblast. However, the underlying molecular mechanisms of LF-induced osteogenic differentiation of mesenchymal stem cells (MSCs) has not been identified. In this study, we tested the hypothesis that the effects of LF on osteogenesis of MSCs required mediation by TGF-ß Receptors and activating TGF-ß signaling pathway. Using siRNA silencing technology, the knockdown of TGF-ß Receptor II (TßRII) could significantly attenuate LF's effect on the proliferation rate and alkaline phosphatase (ALP) activity of MSCs. It indicated that LF induced osteogenic activity that is dependent on TßRII in C3H10T1/2. Subsequently, it was shown that LF activated Smad2. Downregulating TGF-ß Receptor I (TßRI) with SB431542 attenuated the expression of p-Smad2 and p-P38, also the LF-induced the osteogenic activity. Besides, the stimulation by LF on the expression of Osteocalcin (OCN), Osteopontin (OPN), Collagen-2a1 (Col2a1), and Fibroblast Growth Factor 2 (FGF2) were abolished by SB431542. These results confirmed that LF induced osteogenic activity though the TGF-ß canonical and noncanonical signaling pathway. This study provided the first evidence of the signaling mechanisms of LF's effect on osteogenesis in MSCs.


Assuntos
Diferenciação Celular , Lactoferrina/farmacologia , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteocalcina/genética , Osteocalcina/metabolismo , Osteopontina/genética , Osteopontina/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Proteína Smad2/genética , Proteína Smad2/metabolismo
4.
Minerva Med ; 110(5): 419-424, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30938133

RESUMO

BACKGROUND: Osteoarthritis (OA) is a common worldwide disease induced by a wide range of biochemical processes, mainly inflammation and degradation of collagen. The aim of this study, was to describe the effect of a multistrain probiotic (PB) and chondroitin sulfate (CS), administered separately or in combination, on the expression of Ptgs2, Tgfb1 and Col2a1 during monoiodoacetate-induced OA in male rats. METHODS: OA was induced in male rats by injecting monoiodoacetate in right hind knee. Therapeutic groups received 3 mg/kg of CS for 28 days and/or 1.4 g/kg of multistrain PB for 14 days. Knee cartilage were taken 30 days after monoiodoacetate injection. RNA was extracted and the expression of Ptgs2, Tgfb1 and Col2a1 were analyzed using SYBR Green 1-step real-time quantitative polymerase chain reaction. RESULTS: Induction of OA caused an upregulation in Ptgs2, Tgfb1 expression, and downregulation of Col2a1. Separate administration of PB and CS reduced Ptgs2 and Tgfb1 expressions. Their combined administration significantly decreased the expression of these pro-inflammatory cytokines, comparable to controls. Expression of Col2a1 showed similar behavior, with upregulation in therapeutic group with separate administration and the cumulative effects in case of co-administration. CONCLUSIONS: The multistrain PB diet may offer a perspective to improve the standard treatment of OA and, necessitates further investigation with clinical trials.


Assuntos
Sulfatos de Condroitina/uso terapêutico , Colágeno Tipo II/biossíntese , Ciclo-Oxigenase 2/biossíntese , Osteoartrite do Joelho/dietoterapia , Osteoartrite do Joelho/tratamento farmacológico , Probióticos/uso terapêutico , Fator de Crescimento Transformador beta1/biossíntese , Animais , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Sulfatos de Condroitina/administração & dosagem , Colágeno Tipo II/genética , Ciclo-Oxigenase 2/genética , Avaliação Pré-Clínica de Medicamentos , Interações Alimento-Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Ácido Iodoacético/toxicidade , Masculino , Microbiota , Osteoartrite do Joelho/induzido quimicamente , Osteoartrite do Joelho/metabolismo , RNA Mensageiro/biossíntese , Ratos , Fator de Crescimento Transformador beta1/genética
5.
Gene ; 704: 134-141, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30981839

RESUMO

To maintain normal function of cartilage tissue normally, the presence of a sufficient amount of type II collagen and aggrecan is essential, and their synthesis is tightly regulated. Therefore, understanding the mechanisms that control the expression of type II collagen and aggrecan would be useful for understanding gene expression changes in diseases such as osteoarthritis. Recently, we have identified two pairs of enhancer elements, termed E1 and E2 in the type II collagen gene and Ea and Eb in the aggrecan gene. However, their different mechanisms of action remained unclear. Thus, the central aim of this study was to clarify the different transcriptional regulation mediated through each enhancer element. To this end, we established different stable reporter cell lines that express a reporter gene under the control of different enhancer elements using a silent reporter system we previously constructed. Using these cell lines, we found that dexamethasone, forskolin, and trichostatin A affect the gene expression of type II collagen and aggrecan via different enhancer elements. Moreover, we clarified that E1 and E2 enhancer activities are regulated through distinct epigenetic modifications by histone deacetylase 10 and sirtuin 6.


Assuntos
Agrecanas/genética , Colágeno Tipo II/genética , Elementos Facilitadores Genéticos/genética , Epigênese Genética/fisiologia , Agrecanas/metabolismo , Animais , Condrócitos/metabolismo , Condrócitos/patologia , Colágeno Tipo II/metabolismo , Regulação da Expressão Gênica , Histona Desacetilases do Grupo III/metabolismo , Regiões Promotoras Genéticas , Ratos , Sirtuínas/metabolismo , Células Tumorais Cultivadas
6.
Genet Test Mol Biomarkers ; 23(5): 310-315, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30932712

RESUMO

Objective:Heterozygous pathogenic variants in the COL2A1 gene result in several clinical features including impaired skeletal growth, ocular and otolaryngological abnormalities. Missense mutations in the triple helical region of the COL2A1 protein have been associated with lethal spondyloepiphyseal dysplasia (SED). In this study, we aimed to identify the underlying cause of a case of SED congenita (SEDC) in a 27-month-old child. Materials and Methods: A patient who was diagnosed initially with osteochondrodysplasia underwent a detailed clinical and radiological examination to obtain a conclusive diagnosis. The patient did not show any clinical features of hypochondrogenesis. Whole exome sequencing of the COL2A1 gene was carried out to identify the underlying genetic cause of the disorder. Results: Variant annotation and filtration detected a heterozygous missense mutation c.1357G>A (p.G453S) in the exon 21 of the COL2A1 gene of the proband which was confirmed by Sanger sequencing. Neither parent carried the mvariant suggesting this was a new mutation. Conclusion: The COL2A1 mutation (c.1357G>A), identified in this case, results in more mild phenotype than other missense mutations in exon 21 which are known to cause lethal hypochondrogenesis. We showed, for the first time, that a missense mutation (p.G453S) in the triple helical region of the alpha 1 (II) chain of the COL2A1 protein underlies SEDC and is not always lethal.


Assuntos
Colágeno Tipo II/genética , Osteocondrodisplasias/congênito , Colágeno Tipo II/fisiologia , Feminino , Heterozigoto , Humanos , Lactente , Mutação , Mutação de Sentido Incorreto/genética , Osteocondrodisplasias/genética , Osteocondrodisplasias/fisiopatologia , Arábia Saudita , Sequenciamento Completo do Exoma
7.
Mol Med Rep ; 19(5): 4388-4400, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30942441

RESUMO

Osteoarthritis (OA) is an aseptic inflammatory disease which is associated with the stromal cell­derived factor 1/C­X­C chemokine receptor type 4 (SDF­1/CXCR4) axis. Accumulating studies have identified numbers of microRNAs (miRNAs) that serve important roles in the pathogenesis of OA. However, whether and how the inhibition of the SDF­1/CXCR4 axis induces alterations in miRNA expression remains largely unclear. miRNA profiling was performed in OA chondrocytes stimulated with SDF­1 alone, or SDF­1 with the CXCR4 antagonist TN14003 by miRNA microarray. Candidate miRNAs were verified by reverse transcription quantitative polymerase chain reaction. Bioinformatic analyses including target prediction, gene ontology (GO) and pathway analysis were performed to explore the potential functions of candidate miRNAs. Notably, 7 miRNAs (miR­146a­5p, miR­221­3p, miR­126­3p, miR­185­5p, miR­155­5p, miR­124­3p and miR­130a­3p) were significantly differentially expressed. GO analysis indicated that miR­146a­5p and its associated genes were enriched in receptor regulatory activity, nuclear factor­kappa­light­chain­enhancer of activated B cells (NF­κB)­inducing kinase activity, cellular response to interleukin­1, cytokine­cytokine receptor interaction, NF­κB signaling pathway and osteoclast differentiation pathways. CXCR4 was predicted to be a target of miR­146a­5p with high importance. The mRNA and protein levels of key factors involved in cartilage degeneration were measured following manipulation of the expression levels of miR­146a­5p in OA chondrocytes. CXCR4 and MMP­3 levels were negatively associated with miR­146a­5p expression, while the levels of type II collagen and aggrecan were positively associated. These data reveal that TN14003 upregulates miR­146a­5p expression, and also pinpoints a novel role of miR­146a­5p in inhibiting cartilage degeneration by directly targeting the SDF­1/CXCR4 axis.


Assuntos
Quimiocina CXCL12/farmacologia , MicroRNAs/metabolismo , Osteoartrite/patologia , Peptídeos/farmacologia , Regulação para Cima/efeitos dos fármacos , Idoso , Antagomirs/metabolismo , Biomarcadores/metabolismo , Cartilagem/metabolismo , Cartilagem/patologia , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Feminino , Humanos , Masculino , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Osteoartrite/diagnóstico , Osteoartrite/metabolismo , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transdução de Sinais/efeitos dos fármacos
8.
PLoS One ; 14(3): e0212664, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30861010

RESUMO

Osteoarthritis (OA) is a progressive disease associated with cartilage injury and its inherently limited repair capability. Synovium-based cellular constructs (sConstructs) are proposed as possible treatments. Equine sConstructs were produced from decellularized synovium-based extracellular matrix scaffolds (sECM) seeded with synovium-derived mesenchymal stem cells (sMSC), and engineered to express green fluorescent protein (GFP), or bone morphogenetic protein-2 (BMP-2). Survival, distribution, and chondrogenic potential of the sConstructs in vitro and in vivo were assessed. sConstructs in co-culture with chondrocytes increased chondrocyte proliferation, viability, and Col II production, greatest in BMP-2-sConstructs. Chondrocyte presence increased the production of hyaluronic acid (HA), proteoglycan (PG), and BMP-2 by the sConstructs in a positive feedback loop. sECM alone, or GFP- or BMP-2-sConstructs were implanted in synovium adjacent to clinically created full-thickness rat-knee cartilage lesions. At 5 weeks, the lesion area and implants were resected. Gross anatomy, adjacent articulate cartilage growth and subchondral bone repair were scored; and peripheral, central and cartilage lesion measurements taken. For all scores and measurements, sConstruct implants were significantly greater than controls, greatest with the BMP-2-sConstructs. Immunohistochemistry demonstrated migration of endogenous cells into the sECM, with greater cellularity in the constructs with intense positive GFP staining confirming engraftment of implanted sMSC and continued gene expression. In summary, exposing cartilage to sConstructs was chondrogenic in vitro and in vivo, and resulted in substantially increased growth in vivo. This effect was mediated, in part, by soluble ECM and cell factors and upregulation of anabolic growth proteins, such as BMP-2. This work is "proof of concept" that sConstructs surgically implanted adjacent to cartilage damage can significantly improve cartilage and subchondral bone repair, and potentially prevent the progression of OA.


Assuntos
Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Condrogênese , Matriz Extracelular/metabolismo , Articulação do Joelho/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Osteoartrite do Joelho/terapia , Animais , Proteína Morfogenética Óssea 2/biossíntese , Proteína Morfogenética Óssea 2/genética , Cartilagem Articular/patologia , Condrócitos/patologia , Colágeno Tipo II/biossíntese , Colágeno Tipo II/genética , Modelos Animais de Doenças , Matriz Extracelular/patologia , Cavalos , Articulação do Joelho/patologia , Células-Tronco Mesenquimais/patologia , Osteoartrite do Joelho/genética , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/patologia , Ratos , Transdução Genética
9.
Mater Sci Eng C Mater Biol Appl ; 99: 103-111, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30889635

RESUMO

The limited potential of cartilage to regenerate itself has led to development of new strategies and biomaterials for cartilage tissue engineering and regenerative medicine. Although de novo strategies for cartilage repair have been realized, extrudable hydrogels that can be administered in minimally invasive manner while simultaneously supporting chondrogenic differentiation could lead to development of new systems to deliver cells to cartilage lesions. In this work, we explored the suitability of thermo-reversible, extrudable gels derived from carboxylated agarose for maintaining human articular chondrocyte (HAC) phenotype. Towards this objective, we have investigated the impact of hydrogel stiffness and presence of integrin-binding peptide sequence GGGGRGDSP on HAC differentiation potential. We discovered that stiffer hydrogels (5.8 kPa) are more efficient than softer counterparts (0.6 kPa) in promoting chondrogenesis. Interestingly, in GGGGRGDSP modified gels, a synergy between stiffness and RGD signaling led to enhanced expression of chondrogenic related genes (aggrecan, collagen type II and sox9). These findings were also supported by quantitative analysis of sulfated glycosaminoglycans. Since carboxylated agarose are highly suitable as bioink for 3D bioprinting, we propose that extrudable GGGGRGDSP-linked stiff carboxylated agarose as a medium for direct printing of chondrocyte into cartilage lesion.


Assuntos
Condrócitos/metabolismo , Oligopeptídeos/química , Sefarose/química , Adolescente , Biomarcadores/metabolismo , Condrócitos/citologia , Condrogênese/genética , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Hidrogéis/química , Injeções , Masculino , Fenótipo
10.
Int J Mol Sci ; 20(4)2019 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-30781744

RESUMO

During standard expansion culture (i.e., plasma osmolarity, 280 mOsm) human articular chondrocytes dedifferentiate, making them inappropriate for autologous chondrocyte implantation to treat cartilage defects. Increasing the osmolarity of culture media to physiological osmolarity levels of cartilage (i.e., 380 mOsm), increases collagen type II (COL2A1) expression of human articular chondrocytes in vitro, but the underlying molecular mechanism is not fully understood. We hypothesized that TGF-ß superfamily signaling may drive expression of COL2A1 under physiological osmolarity culture conditions. Human articular chondrocytes were cultured in cytokine-free medium of 280 or 380 mOsm with or without siRNA mediated TGF-ß2 knockdown (RNAi). Expression of TGF-ß isoforms, and collagen type II was evaluated by RT-qPCR and immunoblotting. TGF-ß2 protein secretion was evaluated using ELISA and TGF-ß bioactivity was determined using an established reporter assay. Involvement of BMP signaling was investigated by culturing human articular chondrocytes in the presence or absence of BMP inhibitor dorsomorphin and BMP bioactivity was determined using an established reporter assay. Physiological cartilage osmolarity (i.e., physosmolarity) most prominently increased TGF-ß2 mRNA expression and protein secretion as well as TGF-ß bioactivity. Upon TGF-ß2 isoform-specific knockdown, gene expression of chondrocyte marker COL2A1 was induced. TGF-ß2 RNAi under physosmolarity enhanced TGF-ß bioactivity. BMP bioactivity increased upon physosmotic treatment, but was not related to TGF-ß2 RNAi. In contrast, dorsomorphin inhibited COL2A1 mRNA expression in human articular chondrocytes independent of the osmotic condition. Our data suggest a role for TGF-ß superfamily member signaling in physosmolarity-induced mRNA expression of collagen type II. As physosmotic conditions favor the expression of COL2A1 independent of our manipulations, contribution of other metabolic, post-transcriptional or epigenetic factors cannot be excluded in the underlying complex and interdependent regulation of marker gene expression. Dissecting these molecular mechanisms holds potential to further improve future cell-based chondral repair strategies.


Assuntos
Biomarcadores/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Regulação da Expressão Gênica , Humanos , Especificidade de Órgãos , Concentração Osmolar , Isoformas de Proteínas/metabolismo , Interferência de RNA
11.
Medicine (Baltimore) ; 98(1): e13780, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30608389

RESUMO

Spondyloepiphyseal dysplasia congenita (SEDC) is an autosomal dominant disorder, characterized by disproportionate dwarfism with short spine, short neck associated with variable degrees of coxa vara. Cervical cord compression is the most hazardous skeletal deformity in patients with SEDC which requires special attention and management.Ten patients with the clinical and the radiographic phenotypes of spondyloepiphyseal dysplasia congenita have been recognized and the genotype was compatible with single base substitutions, deletions or duplication of part of the COL2A1 gene (6 patients out of ten have been sequenced). Cervical spine radiographs showed apparent atlantoaxial instability in correlation with odontoid hypoplasia or os-odontoideum.Instability of 8 mm or more and or the presence of symptoms of myelopathy were the main indications for surgery. Posterior cervical fusion from the occiput or C1-3, decompression of C1-2 and application of autorib transfer followed by halo vest immobilization have been applied accordingly.Orthopedic management of children with spondyloepiphyseal dysplasia congenita (SEDC) should begin with the cervical spine to avoid serious neurological deficits and or mortality.


Assuntos
Vértebras Cervicais/cirurgia , Descompressão Cirúrgica/métodos , Osteocondrodisplasias/congênito , Compressão da Medula Espinal/cirurgia , Fusão Vertebral/métodos , Adolescente , Vértebra Cervical Áxis/cirurgia , Vértebras Cervicais/diagnóstico por imagem , Criança , Pré-Escolar , Colágeno Tipo II/genética , Feminino , Humanos , Instabilidade Articular/congênito , Instabilidade Articular/cirurgia , Masculino , Osteocondrodisplasias/complicações , Osteocondrodisplasias/genética , Osteocondrodisplasias/cirurgia , Compressão da Medula Espinal/congênito , Doenças da Medula Espinal/congênito , Doenças da Medula Espinal/cirurgia , Doenças da Coluna Vertebral/congênito , Doenças da Coluna Vertebral/cirurgia , Resultado do Tratamento
12.
Mol Med Rep ; 19(3): 2377-2385, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30664218

RESUMO

Leukemia inhibitory factor (LIF) is a multifunctional cytokine. The present study aimed to determine the expression and effects of LIF on nucleus pulposus generation. Degenerated nucleus pulposus samples were obtained from animal models and patients with lumbar intervertebral disc herniation. Degradation scores of intervertebral discs were evaluated via magnetic resonance imaging (MRI) and histology, and the protein expression levels of LIF were detected. Furthermore, cultured primary human degenerated nucleus pulposus cells (DNPCs) were stimulated with various concentrations of recombinant human LIF protein (rhLIF), and aggrecan and collagen type II α1 (COL2α1) protein expression levels were detected by western blotting. In addition, aggrecan expression was determined by toluidine blue staining. The effects of rhLIF on proliferation and apoptosis of DNPCs were evaluated by Cell Counting Kit­8 and flow cytometry, respectively. The results revealed that the degradation scores of intervertebral discs were significantly associated with modeling time, as determined by MRI and histology. In addition, the protein expression levels of LIF were initially increased in patients with lumbar disc herniation and in rabbit models, particularly in the 2­week modeling group; however, its expression decreased with the progression of disc degeneration. Notably, LIF expression in each modeling group was higher than that in the control and 0 week modeling group. The in vitro study revealed that the protein expression levels of aggrecan and COL2α1 were significantly increased in response to rhLIF, in a dose­dependent manner, and statistical differences were identified between the treatment groups and control group. The results of toluidine blue staining were consistent with this finding. Although rhLIF had no effect on proliferation, it inhibited apoptosis of DNPCs in a concentration­dependent manner. In conclusion, LIF was upregulated during the process of intervertebral disc degeneration, and may promote the expression of extracellular matrix components. It may also be hypothesized that LIF acts as a potential protective factor by inhibiting apoptosis of DNPCs without affecting cell proliferation.


Assuntos
Degeneração do Disco Intervertebral/tratamento farmacológico , Fator Inibidor de Leucemia/genética , Núcleo Pulposo/efeitos dos fármacos , Proteínas Recombinantes/administração & dosagem , Agrecanas/genética , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo II/genética , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Disco Intervertebral/diagnóstico por imagem , Disco Intervertebral/metabolismo , Disco Intervertebral/fisiopatologia , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/fisiopatologia , Deslocamento do Disco Intervertebral/tratamento farmacológico , Deslocamento do Disco Intervertebral/genética , Deslocamento do Disco Intervertebral/fisiopatologia , Fator Inibidor de Leucemia/administração & dosagem , Imagem por Ressonância Magnética , Masculino , Núcleo Pulposo/diagnóstico por imagem , Núcleo Pulposo/crescimento & desenvolvimento , Núcleo Pulposo/patologia , Coelhos , Proteínas Recombinantes/genética
14.
Lasers Med Sci ; 34(1): 115-126, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30264177

RESUMO

Tissue engineering aims to take advantage of the ability of undifferentiated stem cells to differentiate into multiple cell types to repair damaged tissue. Photobiomodulation uses either lasers or light-emitting diodes to promote stem cell proliferation and differentiation. The present study aimed to investigate single and dual combinations of laser wavelengths on mesenchymal stem cells (MSCs). MSCs were derived from rabbit iliac bone marrow. One control and eight laser irradiated groups were designated as Infrared (IR, 810 nm), Red (R, 660 nm), Green (G, 532 nm), Blue (B, 485 nm), IR-R, IR-B, R-G, and B-G. Irradiation was repeated daily for 21 days and cell proliferation, osseous, or cartilaginous differentiation was then measured. RT-PCR biomarkers were SOX9, aggrecan, COL 2, and COL 10 expression for cartilage and ALP, COL 1, and osteocalcin expression for bone. Cellular proliferation was increased in all irradiated groups except G. All cartilage markers were significantly increased by IR and IR-B except COL 10 which was suppressed by IR-B combination. ALP expression was highest in R and IR groups during osseous differentiation. ALP was decreased by combinations of IR with B and with R, and also by G alone. R and B-G groups showed stimulated COL 1 expression; however, COL 1 was suppressed in IR-B, IR-R, and G groups. IR significantly increased osteocalcin expression, but in B, B-G, and G groups it was reduced. Cartilage differentiation was stimulated by IR and IR-B laser irradiation. The effects of single or combined laser irradiation were not clear-cut on osseous differentiation. Stimulatory effects on osteogenesis were seen for R and IR lasers, while G laser had inhibitory effects.


Assuntos
Osso e Ossos/citologia , Cartilagem/citologia , Diferenciação Celular/efeitos da radiação , Lasers , Terapia com Luz de Baixa Intensidade , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos da radiação , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Linhagem da Célula/efeitos da radiação , Proliferação de Células/efeitos da radiação , Forma Celular/efeitos da radiação , Células Cultivadas , Condrogênese/genética , Condrogênese/efeitos da radiação , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Regulação da Expressão Gênica/efeitos da radiação , Osteogênese/genética , Osteogênese/efeitos da radiação , Coelhos
15.
BMC Musculoskelet Disord ; 19(1): 449, 2018 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-30579353

RESUMO

BACKGROUND: Osteoarthritis is a degradative joint disease found in humans and commercial swine which can develop from a number of factors, including prior joint trauma. An impact injury model was developed to deliver in vitro loads to disease-free porcine patellae in a model of OA. METHODS: Axial impactions (2000 N normal) and shear impactions (500 N normal with induced shear forces) were delivered to 48 randomly assigned patellae. The patellae were then cultured for 0, 3, 7, or 14 days following the impact. Specimens in the tissue surrounding the loading site were harvested and expression of 18 OA related genes was studied via quantitative PCR. The selected genes were previously identified from published work and fell into four categories: cartilage matrix, degradative enzymes, inflammatory response, and apoptosis. RESULTS: Type II collagen (Col2a1) showed significantly lower expression in shear vs. axial adjacent tissue at day 0 and 7 (fold changes of 0.40 & 0.19, respectively). In addition, higher expression of degradative enzymes and Fas, an apoptosis gene, was observed in the shear specimens. CONCLUSIONS: The results suggest that a more physiologically valid shear load may induce more damage to surrounding articular cartilage than a normal load alone.


Assuntos
Cartilagem Articular/metabolismo , Osteoartrite do Joelho/genética , Patela/metabolismo , Transcriptoma , Animais , Cartilagem Articular/patologia , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Enzimológica da Expressão Gênica , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/patologia , Patela/patologia , Estresse Mecânico , Sus scrofa , Fatores de Tempo , Técnicas de Cultura de Tecidos , Receptor fas/genética , Receptor fas/metabolismo
16.
BMC Med Genet ; 19(1): 212, 2018 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-30541462

RESUMO

BACKGROUND: Dwarfism is a common severe growth disorder, but the etiology is unclear in the majority of cases. Recombinant human growth hormone may be a treatment option, but it has limited efficacy. The currently known laboratory assays do not meet the precision requirements for clinical diagnosis. Here, we have constructed a targeted next-generation sequencing (NGS) panel of selected genes that are suspected to be associated with dwarfism for genetic screening. METHODS: Genetic screening of 91 children with short stature of unknown etiology was performed with the help of the NGS panel. All the coding regions and exon-intron boundaries of 166 genes were included in the panel. To clarify the pathogenicity of these mutations, their clinical data were reviewed and analyzed. RESULTS: The assay identified p.A72G, p.I282V, and p.P491S variants of the PTPN11 gene and a p.I437T variant of the SOS1 gene in 4 cases with Noonan syndrome. A frameshift mutation (p.D2407fs) of the ACAN gene was identified in a case of idiopathic short stature with moderately advanced bone age. A p.R904C variant of the COL2A1 gene was found in a patient, who was accordingly diagnosed with Stickler syndrome. Severe short stature without limb deformity was associated with a p.G11A variant of HOXD13. In addition, we evaluated evidence that a p.D401N variant of the COMP gene may cause multiple epiphyseal dysplasia. CONCLUSIONS: Our findings suggest that syndromes, particularly Noonan syndrome, may be overlooked due to atypical clinical features. This gene panel has been verified to be effective for the rapid screening of genetic etiologies associated with short stature and for guiding precision medicine-based clinical management.


Assuntos
Artrite/genética , Doenças do Tecido Conjuntivo/genética , Nanismo/genética , Perda Auditiva Neurossensorial/genética , Mutação , Síndrome de Noonan/genética , Osteocondrodisplasias/genética , Descolamento Retiniano/genética , Adolescente , Agrecanas/genética , Artrite/diagnóstico , Artrite/etnologia , Artrite/patologia , Grupo com Ancestrais do Continente Asiático , Proteína de Matriz Oligomérica de Cartilagem/genética , Criança , Pré-Escolar , Colágeno Tipo II/genética , Doenças do Tecido Conjuntivo/diagnóstico , Doenças do Tecido Conjuntivo/etnologia , Doenças do Tecido Conjuntivo/patologia , Nanismo/diagnóstico , Nanismo/etnologia , Nanismo/patologia , Feminino , Expressão Gênica , Testes Genéticos/métodos , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/etnologia , Perda Auditiva Neurossensorial/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Homeodomínio/genética , Humanos , Masculino , Síndrome de Noonan/diagnóstico , Síndrome de Noonan/etnologia , Síndrome de Noonan/patologia , Osteocondrodisplasias/diagnóstico , Osteocondrodisplasias/etnologia , Osteocondrodisplasias/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Descolamento Retiniano/diagnóstico , Descolamento Retiniano/etnologia , Descolamento Retiniano/patologia , Proteína SOS1/genética , Fatores de Transcrição/genética
17.
Int J Mol Sci ; 19(12)2018 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-30551562

RESUMO

The neural crest (NC) comprises a multipotent cell population that produces peripheral neurons, cartilage, and smooth muscle cells, among other phenotypes. The participation of Hes1 and Msx1 when expressed in mouse embryonic stem cells (mESCs) undergoing NC differentiation is unexplored. In this work, we generated stable mESCs transfected with constructs encoding chimeric proteins in which the ligand binding domain of glucocorticoid receptor (GR), which is translocated to the nucleus by dexamethasone addition, is fused to either Hes1 (HGR) or Msx1 (MGR), as well as double-transgenic cells (HGR+MGR). These lines continued to express pluripotency markers. Upon NC differentiation, all lines exhibited significantly decreased Sox2 expression and upregulated Sox9, Snai1, and Msx1 expression, indicating NC commitment. Dexamethasone was added to induce nuclear translocation of the chimeric proteins. We found that Collagen IIa transcripts were increased in MGR cells, whereas coactivation of HGR+MGR caused a significant increase in Smooth muscle actin (α-Sma) transcripts. Immunostaining showed that activation in HGR+MGR cells induced higher proportions of ß-TUBULIN III⁺, α-SMA⁺ and COL2A1⁺ cells. These findings indicate that nuclear translocation of MSX-1, alone or in combination with HES-1, produce chondrocyte-like cells, and simultaneous activation of HES-1 and MSX-1 increases the generation of smooth muscle and neuronal cells.


Assuntos
Condrócitos/citologia , Fator de Transcrição MSX1/genética , Células-Tronco Embrionárias Murinas/citologia , Miócitos de Músculo Liso/citologia , Crista Neural/citologia , Receptores de Glucocorticoides/genética , Fatores de Transcrição HES-1/genética , Actinas/genética , Animais , Diferenciação Celular , Núcleo Celular/metabolismo , Células Cultivadas , Condrócitos/metabolismo , Colágeno Tipo II/genética , Dexametasona/farmacologia , Fator de Transcrição MSX1/metabolismo , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/metabolismo , Miócitos de Músculo Liso/metabolismo , Células NIH 3T3 , Crista Neural/metabolismo , Regiões Promotoras Genéticas , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Fatores de Transcrição HES-1/metabolismo
18.
Cell Physiol Biochem ; 51(1): 228-243, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30448827

RESUMO

BACKGROUND/AIMS: Osteoarthritis (OA) is a joint degenerative biomechanical disorder involving immunity, metabolic alterations, inflammation, and cartilage degradation, where chondrocytes play a pivotal role. OA has not effective pharmacological treatments and new therapeutic targets are needed. Adipokines contribute to the low-grade systemic inflammation in OA. Here, we explored novel molecular mechanisms of sodium butyrate (BuNa) in modulating inflammation and chemotaxis in chondrocytes, demonstrating the direct involvement of its G protein-coupled receptor (GPR)-43. METHODS: ATDC5 murine chondrocytes were stimulated with interleukin (IL)-1ß, in the presence or not of BuNa, for 24 h. RT-PCR and Western blot analysis was performed to evaluate the expression of inflammatory mediators and structural proteins. RESULTS: Butyrate reduced the expression of canonic pro-inflammatory mediators (Nos2, COX-2, IL-6), pro-inflammatory adipokines (lipocalin-2 and nesfatin-1) and adhesion molecule (VCAM-1 and ICAM-1) in IL-1ß-stimulated chondrocytes, inhibiting several inflammatory signalling pathways (NFκB, MAPKinase, AMPK-α, PI3K/Akt). Butyrate also reduced metalloproteinase production and limited the loss of type II collagen in IL-1ß-inflamed chondrocytes. The chemoattractant effect of butyrate, after different inflammatory challenges, was revealed by increased annexin (AnxA)1 levels and chemokines expression. The chemoattractant and anti-inflammatory activities of butyrate were completely blunted by GPR43 silencing using RNA interference. CONCLUSION: Taken together, our data suggest the potential application of sodium butyrate as a novel candidate in a multi-target approach for the treatment of chondrocyte inflammation and cartilage degenerative process.


Assuntos
Ácido Butírico/farmacologia , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adipocinas/metabolismo , Animais , Anexina A1/metabolismo , Moléculas de Adesão Celular/metabolismo , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Ciclo-Oxigenase 2/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-1beta/farmacologia , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Camundongos , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/genética
19.
Stem Cell Res Ther ; 9(1): 278, 2018 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-30359317

RESUMO

BACKGROUND: Mesenchymal stem cells (MSCs) isolated from adult tissues (Ad-MSCs) have shown great promise for use in regenerative medicine. However, their poor in vitro expansion capacity and tissue scarcity have been major limitations. In this study, we demonstrate that mouse embryonic stem cells (mESCs) can differentiate into cells with MSC properties. METHODS: Using previously established methods that characterize Ad-MSCs, we analyzed mESC-differentiated fibroblasts (mESC-FBs), including plastic adherence, clonogenic growth, MSC marker expression, tri-lineage differentiation potential, and the capacity to express immunomodulators. RESULTS: Although previously characterized as mESC-differentiated fibroblasts (mESC-FBs), these cells exhibit major properties of Ad-MSCs. However, mESC-FBs also display unique features inherited from ESCs, including robust expansion capacity, senescence resistance, and attenuated innate immunity. In particular, mESC-FBs are insensitive to bacterial endotoxin (lipopolysaccharide, LPS) and do not express LPS-induced inflammatory molecules, in contrast to bone marrow (BM)-MSCs. We further demonstrate that mESC-FBs are resistant to the cytotoxicity associated with inflammatory cytokines, bacterial endotoxins (LPS and heat-killed bacteria), and macrophage-mediated inflammation. CONCLUSIONS: While it remains to be determined how the unique properties of mESC-FBs will affect their immunoregulatory activity under an in vivo condition, our findings demonstrate that ESCs could be used as an alternative source to generate a new class of ESC-MSCs with unique features potentially useful in regenerative medicine.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Tretinoína/farmacologia , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Biomarcadores/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/imunologia , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Colágeno Tipo II/genética , Colágeno Tipo II/imunologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/imunologia , Fibroblastos/citologia , Fibroblastos/imunologia , Expressão Gênica , Humanos , Imunidade Inata , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/imunologia , PPAR gama/genética , PPAR gama/imunologia , Células RAW 264.7 , Medicina Regenerativa/métodos , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/imunologia
20.
Biochem Biophys Res Commun ; 505(3): 692-698, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30292414

RESUMO

Osteoarthritis (OA) is a common joint disease that is regarded as a local inflammatory response caused by joint instability and accompanied by the progressive degeneration of articular cartilage. However, the molecular mechanisms involved in the maintenance of articular cartilage remain a subject of debate and research. This study aims to analyze the roles of long noncoding RNA (lncRNA)CIR and autophagy in cartilages and determine their overall contribution to the degradation of extracellular matrix. Patients with OA possessed high levels of lncRNA-CIR and MMP3 and low level of COL2A1. The levels of autophagy-related proteins, including LC3BI/II and beclin-1, increased from 12 h to 48 h. The use of si-lncRNA-CIR reversed the trend compared with that in the OA group. The negative effect of lncRNA-CIR was assessed in vivo by establishing a model of surgically induced OA. Moreover, si-lncRNA-CIR-treated joints exhibited fewer OA changes than saline-treated joints. Results were confirmed by histopathological grading of the models by using the Osteoarthritis Research Society International Scoring System and the outcomes of immunohistochemistry for LC3B-II and MMP-3. Overall, lncRNA-CIR played a negative role in the OA process by activating autophagy.


Assuntos
Autofagia/genética , Cartilagem Articular/metabolismo , Osteoartrite/genética , RNA Longo não Codificante/genética , Adulto , Animais , Proteína Beclina-1/metabolismo , Cartilagem Articular/patologia , Células Cultivadas , Condrócitos/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Osteoartrite/metabolismo , Ratos Sprague-Dawley
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