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1.
Biochem Biophys Res Commun ; 663: 61-70, 2023 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-37119767

RESUMO

Articular cartilage (AC) is most susceptible to degeneration in knee osteoarthritis (OA); however, the existing treatments for OA do not target the core link of the pathogenesis-"decreased tissue cell function activity and extracellular matrix (ECM) metabolic disorders" for effective intervention. iMSC hold lower heterogeneity and great promise in biological research and clinical applications. Rps6ka2 may play an important role in the iMSC to treat OA. In this study, the CRISPR/Cas9 gene editing Rps6ka2-/- iMSC were obtained. Effect of Rps6ka2 on iMSC proliferation and chondrogenic differentiation was evaluated in vitro. An OA model was constructed in mice by surgical destabilization of medial meniscus (DMM). The Rps6ka2-/- iMSC and iMSC were injected into the articular cavity twice-weekly for 8 weeks. In vitro experiments showed that Rps6ka2 could promote iMSC proliferation and chondrogenic differentiation. In vivo results further confirmed that Rps6ka2 could improve iMSC viability to promote ECM production to attenuate OA in mice.


Assuntos
Cartilagem Articular , Osteoartrite do Joelho , Camundongos , Animais , Osteoartrite do Joelho/genética , Osteoartrite do Joelho/terapia , Osteoartrite do Joelho/metabolismo , Cartilagem Articular/metabolismo , Diferenciação Celular/genética , Matriz Extracelular , Condrócitos/metabolismo , Modelos Animais de Doenças
2.
Biochem Biophys Res Commun ; 570: 199-205, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34298323

RESUMO

Osteoarthritis (OA) is the most common joint disease worldwide; however, disease-modifying treatments are lacking because of the complicated pathological mechanisms. As a breakthrough, aberrant activation of transforming growth factor-ß 1 (TGF-ß1)in subchondral bone has been confirmed as an essential pathomechanism for OA progression, and has become a potential therapeutic target. In addition to R&D on neutralizing antibodies, small-molecule antagonists and chemical medicines, native antagonists of TGF-ß1 could be exploited as another promising approach. Noggin (NOG) is an antagonist of bone morphogenetic proteins (BMPs) and was reported to effectively attenuate OA by protecting cartilage and preventing pathological subchondral bone remodeling. However, the underlying mechanisms have not been fully clarified. We first detected the distribution of NOG in knee joints of an OA mouse model, which showed upregulation at early stage of OA but downregulation later in the subchondral bone and no significant change in the articular cartilage. Furthermore, the interaction between NOG and TGF-ß1 was verified, which in turn suppressed the downstream SMAD2/3 activity of TGF-ß1. Moreover, the proliferation and chondrogenesis of mesenchymal stem cells (MSCs) were not significantly influenced by NOG. Taken together, the results showed that NOG antagonized TGF-ß1 but did not repress MSC proliferation and chondrogenesis; thus, it seems promising for OA treatment.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Transporte/metabolismo , Osteoartrite/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Animais , Cartilagem Articular/patologia , Proliferação de Células , Condrogênese , Modelos Animais de Doenças , Progressão da Doença , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Osteoartrite/patologia , Ligação Proteica , Fator de Crescimento Transformador beta1/metabolismo
3.
BMC Mol Cell Biol ; 23(1): 32, 2022 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-35883033

RESUMO

BACKGROUND: The single-cell platform provided revolutionary way to study cellular biology. Technologically, a sophistic protocol of isolating qualified single cells would be key to deliver to single-cell platform, which requires high cell viability, high cell yield and low content of cell aggregates or doublets. For musculoskeletal tissues, like bone, cartilage, nucleus pulposus and tendons, as well as their pathological state, which are tense and dense, it's full of challenge to efficiently and rapidly prepare qualified single-cell suspension. Conventionally, enzymatic dissociation methods were wildly used but lack of quality control. In the present study, we designed the rapid cycling enzymatic processing method using tissue-specific enzyme cocktail to treat different human pathological musculoskeletal tissues, including degenerated nucleus pulposus (NP), ossifying posterior longitudinal ligament (OPLL) and knee articular cartilage (AC) with osteoarthritis aiming to rapidly and efficiently harvest qualified single-cell suspensions for single-cell RNA-sequencing (scRNA-seq). RESULTS: We harvested highly qualified single-cell suspensions from NP and OPLL with sufficient cell numbers and high cell viability using the rapid cycling enzymatic processing method, which significantly increased the cell viability compared with the conventional long-time continuous digestion group (P < 0.05). Bioanalyzer trace showed expected cDNA size distribution of the scRNA-seq library and a clear separation of cellular barcodes from background partitions were verified by the barcode-rank plot after sequencing. T-SNE visualization revealed highly heterogeneous cell subsets in NP and OPLL. Unfortunately, we failed to obtain eligible samples from articular cartilage due to low cell viability and excessive cell aggregates and doublets. CONCLUSIONS: In conclusion, using the rapid cycling enzymatic processing method, we provided thorough protocols for preparing single-cell suspensions from human musculoskeletal tissues, which was timesaving, efficient and protective to cell viability. The strategy would greatly guarantee the cell heterogeneity, which is critical for scRNA-seq data analysis. The protocol to treat human OA articular cartilage should be further improved.


Assuntos
Cartilagem Articular , Separação Celular/métodos , Sobrevivência Celular , Humanos , Análise de Sequência de RNA/métodos , Suspensões
4.
Theranostics ; 10(15): 6915-6927, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32550912

RESUMO

Rationale: Cartilage stem/progenitor cells (CSPC) are a promising cellular source to promote endogenous cartilage regeneration in osteoarthritis (OA). Our previous work indicates that ribosomal s6 kinase 3 (RSK-3) is a target of 4-aminobiphenyl, a chemical enhancing CSPC-mediated cartilage repair in OA. However, the primary function and mechanism of RSK-3 in CSPC-mediated cartilage pathobiology remain undefined. Methods: We systematically assessed the association of RSK-3 with OA in three mouse strains with varying susceptibility to OA (MRL/MpJ>CBA>STR/Ort), and also RSK-3-/- mice. Bioinformatic analysis was used to identify the possible mechanism of RSK-3 affecting CSPC, which was further verified in OA mice and CSPC with varying RSK-3 expression induced by chemicals or gene modification. Results: We demonstrated that the level of RSK-3 in cartilage was positively correlated with cartilage repair capacities in three mouse strains (MRL/MpJ>CBA>STR/Ort). Enhanced RSK-3 expression by 4-aminobiphenyl markedly attenuated cartilage injury in OA mice and inhibition or deficiency of RSK-3 expression, on the other hand, significantly aggravated cartilage damage. Transcriptional profiling of CSPC from mice suggested the potential role of RSK-3 in modulating cell proliferation. It was further shown that the in vivo and in vitro manipulation of the RSK-3 expression indeed affected the CSPC proliferation. Mechanistically, ribosomal protein S6 (rpS6) was activated by RSK-3 to accelerate CSPC growth. Conclusion: RSK-3 is identified as a key regulator to enhance cartilage repair, at least partly by regulating the functionality of the cartilage-resident stem/progenitor cells.


Assuntos
Cartilagem/citologia , Condrócitos/citologia , Osteoartrite/terapia , Regeneração , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteína S6 Ribossômica/metabolismo , Células-Tronco/citologia , Compostos de Aminobifenil/farmacologia , Animais , Carcinógenos/farmacologia , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Endogâmicos MRL lpr , Camundongos Knockout , Osteoartrite/metabolismo , Osteoartrite/patologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo
5.
Theranostics ; 9(24): 7108-7121, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31695756

RESUMO

Rationale The small molecule Kartogenin (KGN) promotes cartilage regeneration in osteoarthritis (OA) by activating stem cells differentiation, but its pharmacological mode-of-action remains unclear. KGN can be cleaved into 4-aminobiphenyl (4-ABP) and phthalic acid (PA) following enzymolysis of an amide bond. Therefore, this study investigated whether 4-ABP or PA exerted the same action as KGN. Methods KGN, 4-ABP and PA were analyzed in cartilage of mice after oral, intravenous or intra-articular administration of KGN by liquid chromatography-mass spectrometry method. Their effect on proliferation and chondrogenic differentiation of mesenchymal stem cells (MSC) was evaluated in vitro. Furthermore, their effect on cartilage preservation was tested in mice OA model induced by destabilization of medial meniscus. OA severity was quantified using OARSI histological scoring. Transcriptional analysis was used to find the possible targets of the chemicals, which were further validated. Results We demonstrated that while oral or intra-articular KGN delivery effectively ameliorated OA phenotypes in mice, only 4-ABP was detectable in cartilage. 4-ABP could induce chondrogenic differentiation and proliferation of MSC in vitro and promote cartilage repair in OA mouse models mainly by increasing the number of CD44+/CD105+ stem-cell and prevention of matrix loss. These effect of 4-ABP was stronger than that of KGN. Transcriptional profiling of 4-ABP-stimulated MSC suggested that RPS6KA2 and the PI3K-Akt pathway were 4-ABP targets; 4-ABP could activate the PI3K-Akt pathway to promote MSC proliferation and repair OA injury, which was blocked in RPS6KA2-knockdown MSC or RPS6KA2-deficient mice.Conclusion 4-ABP bio-distribution in cartilage promotes proliferation and chondrogenic differentiation of MSC, and repairs osteoarthritic lesions via PI3K-Akt pathway activation.


Assuntos
Compostos de Aminobifenil/metabolismo , Anilidas/metabolismo , Cartilagem/metabolismo , Ácidos Ftálicos/metabolismo , Regeneração , Administração Oral , Anilidas/administração & dosagem , Anilidas/farmacologia , Animais , Antígenos CD/metabolismo , Cartilagem/efeitos dos fármacos , Cartilagem/lesões , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Humanos , Hidrólise , Masculino , Menisco/efeitos dos fármacos , Menisco/patologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Ácidos Ftálicos/administração & dosagem , Ácidos Ftálicos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regeneração/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Distribuição Tecidual/efeitos dos fármacos
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