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OBJECTIVE: The temporomandibular joint (TMJ) disc cushions intraarticular stress during mandibular movements. While mechanical overloading is related to cartilage degeneration, the pathogenesis of TMJ disc degeneration is unclear. Here, we determined the regulatory role of mechanoinductive transient receptor potential vanilloid 4 (TRPV4) in mechanical overload-induced TMJ disc degeneration. METHODS: We explored the effect of mechanical overload on the TMJ discs in a rat occlusal interference model in vivo, and by applying sustained compressive force in vitro. TRPV4 inhibition was delivered by small interfering RNA or GSK2193874; TRPV4 activation was delivered by GSK1016790A. The protective effect of TRPV4 inhibition was validated in the rat occlusal interference model. RESULTS: Occlusal interference induced TMJ disc degeneration with enhanced extracellular matrix degradation in vivo and mechanical overload promoted inflammatory responses in the TMJ disc cells via Ca2+ influx with significantly upregulated TRPV4. TRPV4 inhibition reversed mechanical overload-induced inflammatory responses; TRPV4 activation simulated mechanical overload-induced inflammatory responses. Moreover, TRPV4 inhibition alleviated TMJ disc degeneration in the rat occlusal interference model. CONCLUSION: Our findings suggest TRPV4 plays a pivotal role in the pathogenesis of mechanical overload-induced TMJ disc degeneration and may be a promising target for the treatment of degenerative changes of the TMJ disc.
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Cellular senescence is implicated in several lines of aging-related disorders. However, the potential molecular mechanisms by which cellular senescence modulates age-related pathologies remain largely unexplored. Herein, we report that the density of sympathetic fibers (SFs) is significantly elevated in naturally aged mouse tissues and human colon adenoma tissues compared to the SFs densities in the corresponding young mouse tissues and human non-lesion colon tissues. A dorsal root ganglion (DRG)-human diploid fibroblast coculture assay revealed that senescent cells promote the outgrowth of SFs, indicating that the senescent cells induce recruitment of SFs in vitro. Additionally, subcutaneous transplantation of 2BS fibroblasts in nude mice shows that transplanted senescent 2BS fibroblasts promote SFs infiltration. Intra-articular senolytic molecular injection can reduce SFs density and inhibit SFs infiltration caused by senescent cells in osteoarthritis (OA), suggesting senescent cells promote the infiltration of SFs in vivo in aged tissues. Notably, the elevated level of SFs contributes to impaired cognitive function in naturally aged mice, which can be reversed by treatment with propranolol hydrochloride, a non-selective ß receptor blocker that inhibits sympathetic nerve activity (SNA) by blocking non-selective ß receptors. Additionally, 6-hydroxydopamine (6-OHDA)-induced sympathectomy improved hepatic sympathetic overactivity mediated hepatic steatosis in high fat diet (HFD)-fed APOE knockout mice (APOE-/- mice) by reducing hepatic SNA. Taken together, this study concludes that senescent cell-secreted netrin-1 mediated SFs outgrowth and infiltration, which contributes to aging-related disorders, suggesting that clearing senescent cells or inhibiting SNA is a promising therapeutic strategy for improving sympathetic nervous system (SNS) hyperactivity-induced aging-related pathologies.
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Circular RNAs (CircRNAs) are a novel subset of non-coding RNA widely present in eukaryotes that play a central role in physiological and pathological conditions. Accumulating evidence has indicated that CircRNAs participated in modulating tumorigenesis by acting as a competing endogenous RNA (CeRNA). However, the roles and functions of CircRNAs in cellular senescence and aging of organisms remain largely obscure. We performed whole transcriptome sequencing to compare the expression patterns of circular RNAs in young and prematurely senescent human diploid fibroblast 2BS cells, and identified senescence-associated circRNAs (SAC-RNAs). Among these SAC-RNAs, we observed the significantly downregulated expression of CircRNAs originating from exons 6 and 7 circularization of the cyclin B1 gene (CCNB1), termed CircCCNB1. Reduced CircCCNB1 expression triggered senescence in young 2BS cells, as measured by increased senescence associated-beta-galactosidase (SA-ß-gal) activity, enhanced expression of cyclin-dependent kinase inhibitor 1A (CDKN1A)/P21 and tumor protein 53 (TP53) expression, and reduced cell proliferation. Mechanistically, reduced CircCCNB1 level inhibited cyclin E2 (CCNE2) expression by modulating micro RNA (miR)-449a activity, which repressed cellular proliferation. Our data suggested that CircCCNB1may serve as a sponge against miR-449a to delay cellular senescence by targeting CCNE2. Targeting CircCCNB1 may represent a promising strategy for aging and age-related disease interventions. Furthermore, we also identified and characterized several kinds of the CircCCNB1-binding proteins (CBPs), which may contribute to the degradation of CircCCNB1.
Assuntos
Senescência Celular/genética , Ciclinas/genética , Regulação da Expressão Gênica/genética , MicroRNAs/genética , RNA Circular/genética , Linhagem Celular , Ciclina B1/genética , Genes cdc/genética , HumanosRESUMO
Long non-coding RNAs (lncRNAs) have recently emerged as key players in many physiologic and pathologic processes. Although many lncRNAs have been identified, few lncRNAs have been characterized functionally in aging. In this study, we used human fibroblast cells to investigate genome-wide lncRNA expression during cellular senescence. We identified 968 down-regulated lncRNAs and 899 up-regulated lncRNAs in senescent cells compared with young cells. Among these lncRNAs, we characterized a senescence-associated lncRNA (SALNR), whose expression was reduced during cellular senescence and in premalignant colon adenomas. Overexpression of SALNR delayed cellular senescence in fibroblast cells. Furthermore, we found that SALNR interacts with NF90 (nuclear factor of activated T-cells, 90 kDa), an RNA-binding protein suppressing miRNA biogenesis. We demonstrated that NF90 is a SALNR downstream target, whose inhibition led to premature senescence and enhanced expressions of senescence-associated miRNAs. Moreover, our data showed that Ras-induced stress promotes NF90 nucleolus translocation and suppresses its ability to suppress senescence-associated miRNA biogenesis, which could be rescued by SALNR overexpression. These data suggest that lncRNA SALNR modulates cellular senescence at least partly through changing NF90 activity.
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Senescência Celular/genética , Proteínas do Fator Nuclear 90/fisiologia , Oncogenes , RNA Longo não Codificante/genética , Nucléolo Celular/metabolismo , Células Cultivadas , Estudo de Associação Genômica Ampla , Humanos , Transporte ProteicoRESUMO
Cellular senescence is a stable state of proliferative arrest that provides a barrier against malignant transformation and contributes to the antitumor activity of certain chemotherapies. Unexpectedly, we found that the expression of proto-oncogene PIM-1, which can promote tumorigenesis, is induced at transcriptional level during senescence. Inhibition of PIM-1 alleviated both replicative and oncogene-induced senescence. Conversely, ectopic expression of PIM-1 resulted in premature senescence. We also revealed that PIM-1 interacts with and phosphorylates heterochromatin protein 1γ (HP1γ) on Ser93. This PIM-1-mediated HP1γ phosphorylation enhanced HP1γ's capacity to bind to H3K9me3, resulting in heterochromatin formation and suppression of proliferative genes, such as CCNA2 and PCNA. Analysis of the mechanism underlying the up-regulation of PIM-1 expression during senescence demonstrated that IL-6, a critical regulator of cellular senescence, is responsible for PIM-1 induction. Our study demonstrated that PIM-1 is a key component of the senescence machinery that contributes to heterochromatin formation. More importantly, we demonstrated that PIM-1 is also a direct target of IL-6/STAT3 signaling and mediates cytokine-induced cellular senescence.
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Heterocromatina/metabolismo , Interleucina-6/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Senescência Celular/fisiologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Fosforilação , Proto-Oncogene Mas , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Ativação Transcricional , Regulação para CimaRESUMO
DNA damage may regulate microRNA (miRNA) biosynthesis at the levels of miRNA transcription, processing and maturation. Although involvement of E2F1 in the regulation of miRNA gene activation in response to DNA damage has been documented, little is known about the role of E2F1 in miRNA processing. In this study we demonstrate that E2F1 enhances miR-630 biosynthesis under cisplatin (CIS) exposure through promoting DROSHA-mediated pri-miR-630 processing. Northern blot and RT-qPCR revealed that CIS exposure caused not only an increase in pri-miR-630 but also much more increase in pre-miR-630 and mature miR-630. The increases in pri-miR-630 and pre-miR-630 expression in unmatched proportion indicated that primary transcript processing was involved in CIS-stimulated miR-630 biosynthesis. Furthermore, combination of reporter enzyme assay with mutation and over-expression of E2F1 showed that induction of DROSHA promoted miR-630 expression, in which CIS-induced E2F1 activated DROSHA gene expression by recognizing and binding two E2F1 sites at the positions -214/-207 and -167/-160 of the DROSHA promoter. The increased binding of E2F1 to the DROSHA promoter in CIS-exposed cells was further evidenced by chromatin immunoprecipitation assay. Together, E2F1-regulated DROSHA promotes pri-miR-630 processing, thereby, contributes to CIS-stimulated miR-630 expression. The involvement of E2F1-dependent DROSHA activation in pri-miRNA processing under DNA damage stress will provide further insight into the regulation of miRNA biosynthesis. These data also give us a deeper understanding of E2F1 role in response to DNA damage.
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Cisplatino/farmacologia , Fator de Transcrição E2F1/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , MicroRNAs/biossíntese , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Ribonuclease III/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , HumanosRESUMO
Akt/protein kinase B is a pivotal component downstream of phosphatidylinositol 3-kinase (PI3K) pathway, whose activity regulates the balance between cell survival and apoptosis. Phosphorylation of Akt occurs at two key sites either at Thr308 site in the activation loop or at Ser473 site in the hydrophobic motif. The phosphorylated form of Akt (pAkt) is activated to promote cell survival. The mechanisms of pAkt dephosphorylation and how the signal transduction of Akt pathway is terminated are still largely unknown. In this study, we identified a novel protein phosphatase CSTP1(complete s transactivated protein 1), which interacts and dephosphorylates Akt specifically at Ser473 site in vivo and in vitro, blocks cell cycle progression and promotes cell apoptosis. The effects of CSTP1 on cell survival and cell cycle were abrogated by depletion of phosphatase domain of CSTP1 or by expression of a constitutively active form of Akt (S473D), suggesting Ser473 site of Akt as a primary cellular target of CSTP1. Expression profile analysis showed that CSTP1 expression is selectively down-regulated in non-invasive bladder cancer tissues and over-expression of CSTP1 suppressed the size of tumors in nude mice. Kaplan-Meier curves revealed that decreased expression of CSTP1 implicated significantly reduced recurrence-free survival in patients suffered from non-invasive bladder cancers.
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Apoptose/fisiologia , Calcineurina/fisiologia , Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina/metabolismo , Neoplasias da Bexiga Urinária/patologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Calcineurina/genética , Primers do DNA , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Invasividade Neoplásica , Fosforilação , Proteínas Proto-Oncogênicas c-akt/química , Interferência de RNA , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias da Bexiga Urinária/enzimologiaRESUMO
BACKGROUND: Candida albicans is a human commensal that is also responsible for chronic gastritis and peptic ulcerous disease. Little is known about the genetic profiles of the C. albicans strains in the digestive tract of dyspeptic patients. The aim of this study was to evaluate the prevalence, diversity, and genetic profiles among C. albicans isolates recovered from natural colonization of the digestive tract in the dyspeptic patients. METHODS AND FINDINGS: Oral swab samples (nâ=â111) and gastric mucosa samples (nâ=â102) were obtained from a group of patients who presented dyspeptic symptoms or ulcer complaints. Oral swab samples (nâ=â162) were also obtained from healthy volunteers. C. albicans isolates were characterized and analyzed by multilocus sequence typing. The prevalence of Candida spp. in the oral samples was not significantly different between the dyspeptic group and the healthy group (36.0%, 40/111 vs. 29.6%, 48/162; P > 0.05). However, there were significant differences between the groups in the distribution of species isolated and the genotypes of the C. albicans isolates. C. albicans was isolated from 97.8% of the Candida-positive subjects in the dyspeptic group, but from only 56.3% in the healthy group (P < 0.001). DST1593 was the dominant C. albicans genotype from the digestive tract of the dyspeptic group (60%, 27/45), but not the healthy group (14.8%, 4/27) (P < 0.001). CONCLUSIONS: Our data suggest a possible link between particular C. albicans strain genotypes and the host microenvironment. Positivity for particular C. albicans genotypes could signify susceptibility to dyspepsia.
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Candida albicans/genética , Candidíase/microbiologia , Dispepsia/microbiologia , Mucosa Gástrica/microbiologia , Mucosa Bucal/microbiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Candida albicans/isolamento & purificação , Candidíase/epidemiologia , Estudos de Casos e Controles , Suscetibilidade a Doenças , Dispepsia/epidemiologia , Feminino , Variação Genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Tipagem de Sequências Multilocus , Técnicas de Tipagem Micológica , Filogenia , Prevalência , Adulto JovemRESUMO
Vitamin D(3) up-regulated protein 1 (VDUP1) plays multifunctional roles in diverse cellular responses, particularly in its relation to proliferation, apoptosis, differentiation, and diseases such as cancer and stress-related diseases. In this study, we demonstrated that VDUP1 was up-regulated during the senescence process. Our results showed that overexpression of VDUP1 in young cells caused typical signs of senescence. We also found that VDUP1 knockdown delayed the onset of Ras-induced cellular senescence. Subsequently, we found that FOXO3A, whose activity increased in senescent cells, transcriptionally up-regulates VDUP1 expression and miR-17-5p, whose expression decreased in senescent cells, directly interacted with the 3'-untranslated region of VDUP1 transcripts, and destabilized VDUP1 mRNA in young cells. These results indicated that VDUP1 expression was regulated by FOXO3A at the transcriptional level and by miR-17-5p at the post-transcriptional levels during the senescence process.
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Regiões 3' não Traduzidas/fisiologia , Proteínas de Transporte/biossíntese , Senescência Celular/fisiologia , Fatores de Transcrição Forkhead/metabolismo , MicroRNAs/metabolismo , Estabilidade de RNA/fisiologia , Transcrição Gênica/fisiologia , Regulação para Cima/fisiologia , Proteínas de Transporte/genética , Células Cultivadas , Fibroblastos , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Técnicas de Silenciamento de Genes , Humanos , Masculino , MicroRNAs/genética , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
This study compared tankyrase 1 expression and autophagy quantity between erectile dysfunction (ED) and non-ED rats' corpus cavernosum smooth muscle cells (CSMCs). This study aslo explored the effect and possible mechanism of tankyrase 1 on autophagy and cell proliferation in ageing ED rats' CSMCs. The intracavernous pressure and mean systemic arterial pressure were measured to investigate erectile function so that eight 24-month-old ED and eight 8-month-old male Wistar rats were chosen respectively. The rat CSMCs were isolated and cultured by enzyme digestion, in which tankyrase 1 expression and autophagy quantity were compared. Tankyrase 1 overexpression was induced with plasmid transfection by Lipofectamine. The effect of tankyrase 1 overexpression on proliferation, autophagy and mTOR pathway in 24-month-old ED rats' CSMCs was measured by the cell growth curve in MTT assay, cell cycle analysis in flow cytometry (FCM), key protein expression in Western blot, autophagy quantity in transmission electron microscopy, monodansylcadaverine staining and GFP-LC3 fluorescence. The primary CSMCs were confirmed by immunofluorescence, and the purity was 99.1% in FCM. Compared with that of 8-month-old rats, tankyrase 1 expression and autophagy quantity significantly decreased in 24-month-old ED rats' primary CSMCs (P < 0.01). Tankyrase 1 overexpression significantly increased the growth rate (P < 0.05) and increased the S phase of cell cycle (P < 0.01). The autophagosome quantity was remarkably increased (P < 0.01), LC3-I/II and Beclin 1 were upregulated (P < 0.01 and P < 0.05), and p-p70S6K (Thr(389)) was downregulated in 24-month-old ED rat CSMCs (P < 0.05). In conclusion, Tankyrase 1 and autophagy decrease in the CSMCs from aging rats with ED, and tankyrase 1 may have a positive effect on proliferation by enhancing autophagy and regulating the mTOR signalling pathway.
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Disfunção Erétil/fisiopatologia , Pênis/fisiopatologia , Tanquirases/fisiologia , Envelhecimento/fisiologia , Animais , Autofagia/fisiologia , Modelos Animais de Doenças , Masculino , Proteínas de Membrana/fisiologia , Miócitos de Músculo Liso/fisiologia , Ratos , Ratos Wistar , Transdução de Sinais/fisiologia , Tanquirases/metabolismoRESUMO
AIM: To study the influencing factors of the increased expression of the controlling insulin growth factors binding protein-3(IGFBP-3)in the senescent cells. METHODS: Northern blot was used to show of the differential expression of the IGFBP-3 gene in the young and senescent 2BS cells; The size of 2 kb human IGFBP-3 upstream sequence including the series of the 5'-UTR area was amplified by PCR, and four groups of IGFBP-3 promoter fragments of different lengths were obtained by enzyme digestion. The Effectene Transfection Reagent (Qiagen) kit was used to transfect the fragments into the young and senescent cells. The promoting activity of several groups of constructing gene fragments were evaluated. The area of the controlling transfection activity was determined. The enhancer element in the activity area was ascertained by superimposing the oligonucleotide gel blocking experiment. RESULTS: Compared with the young 2BS cells, the expression of the IGFBP-3 gene in the senescent 2BS cells was enhanced. There was a protein binding in the fragment site from site +59 to -58 of the IGFBP-3 enhancer. 5'-ccagcctgccaagcagcgtgccccggttgc-3' was the enhancer element of IGFBP-3. CONCLUSION: In the 30 bp fragment from site -37 to -8 of the IGFBP-3 gene upstream, there is a new IGFBP-3 enhancer element IEE, which plays a controlling role in the expression of IGFBP-3.
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Senescência Celular/genética , Perfilação da Expressão Gênica , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Sequência de Bases , Northern Blotting , Linhagem Celular , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Luciferases/genética , Luciferases/metabolismo , Mutação , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Fatores de Tempo , TransfecçãoRESUMO
BACKGROUND: Remodeling of the anterior cruciate ligament (ACL) graft usually takes longer than expected. Gene therapy offers a radical different approach to remodeling of the graft. In this study, the internal ribosome entry site (IRES) sequence was used to construct a new recombinant adenovirus which permits co-expression of transforming growth factor-beta1 (TGFbeta1) and vascular endothelial growth factor 165 (VEGF165) genes (named Ad-VEGF165-IRES-TGFbeta1). We investigated the effects of the new adenovirus on the migration of and matrix synthesis by ACL fibroblasts. METHODS: Adenoviral vector containing TGFbeta1 and VEGF165 genes was constructed. ACL fibroblasts were obtained from New Zealand white rabbits. After ACL fibroblasts were exposed to Ad-VEGF165-IRES-TGFbeta1, the expression of VEGF165 and TGFbeta1 proteins were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis. Bioassay of VEGF165 and TGFbeta1 proteins were assessed by Western blotting analysis. Proliferation and migration of ACL fibroblasts were assessed by in vitro wound closure assay. Gene expression of collagen type I, collagen type III, and fibronectin mRNA among matrix markers were assessed by real-time PCR. RESULTS: The results showed the successful construction of a recombinant co-expression adenovirus vector containing TGFbeta1 and VEGF165 genes. Co-expression of TGFbeta1 and VEGF165 can induce relatively rapid and continuous proliferation of ACL fibroblasts and high gene expression of collagen type I, collagen type III, and fibronectin mRNA among matrix markers. CONCLUSION: Co-expression of TGFbeta1 and VEGF165 genes has more powerful and efficient effects on the migration of and matrix synthesis by ACL fibroblasts.
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Ligamento Cruzado Anterior/metabolismo , Terapia Genética , Fator de Crescimento Transformador beta1/genética , Fator A de Crescimento do Endotélio Vascular/genética , Adenoviridae/genética , Animais , Ligamento Cruzado Anterior/citologia , Movimento Celular , Células Cultivadas , Colágeno/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/fisiologia , Fibronectinas/genética , Vetores Genéticos , Humanos , Coelhos , CicatrizaçãoRESUMO
BACKGROUND: Osteoarthritis (OA) is a chronic and incurable disease, lacking effective treatment. Gene therapy offers a radical different approach to the treatment of arthritis. Even though the etiology of OA remains unclear, there is now considerable evidence to suggest that interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) are the main mediators in the pathogenesis of OA. The goal of this study was to determine the efficacy of local expression of interleukin-1 receptor antagonist (IL-1Ra) and soluble tumor necrosis factor-alpha receptor type I (sTNF-RI) by direct adenoviral-mediated intra-articular gene delivery in the rabbit model of osteoarthritis. METHODS: Adenoviral vectors containing IL-1Ra or sTNF-RI genes were constructed. OA was induced in both hind knees of 12 New Zealand white rabbits by the excision of the medial collateral ligament plus medial meniscectomy. Five days after surgery, approximately 1 x 10(8) plaque-forming units (pfu) of adenovirus were injected into the joint space of the knee through the patellar tendon. A total of 12 operated rabbits were divided into four groups. Three experimental rabbit groups received 1 x 10(8) pfu of adenovirus encoding either IL-1Ra (3 rabbits), sTNF-RI (3 rabbits) or IL-1Ra and sTNF-RI in combination (3 rabbits), into both knee joints respectively. An inflamed control group of 3 rabbits received approximately 1 x 10(8) pfu of Ad-GFP into both joints. Three days after injection of the adenovirus, both knees of each rabbit were lavaged with 1 ml of saline solution through the patellar tendon. At day 7, the rabbits were sacrificed, and the knees were lavaged, dissected and analyzed for effects of transgene expression. Levels of IL-1Ra and sTNF-RI expression in recovered lavage fluids were measured using a cytokine ELISA kit. Cartilage from the lesion areas of medial femoral condyle and synovium were fixed, embedded, sectioned and stained with hematoxylin and eosin (cartilage and synovium) and toluidine blue (cartilage). The samples were examined by light microscopy and quantitatively evaluated. RESULTS: Intra-articular delivery of IL-1Ra resulted in a significant inhibition of cartilage degradation, but did not affect synovial changes. In contrast, rabbit knee joints receiving sTNF-RI alone showed no detectable reduction in cartilage degradation. However, double gene transfer of IL-1Ra and sTNF-RI resulted in a higher suppression of the cartilage degradation and an observable reduction in synovitis. These data add to and confirm that IL-1Ra has good chondroprotective properties, but TNF-alpha blockade has little effect on joint destruction. CONCLUSION: The enhanced therapeutic effects of both antagonists in combination suggest inhibition of multiple inflammatory cytokines may be more efficacious than blockade of either cytokine alone in treating OA.
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Adenoviridae/genética , Terapia Genética/métodos , Osteoartrite/terapia , Animais , Artrite Experimental/genética , Artrite Experimental/terapia , Cartilagem/metabolismo , Cartilagem/patologia , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Linhagem Celular , Células Cultivadas , Vetores Genéticos/genética , Humanos , Proteína Antagonista do Receptor de Interleucina 1 , Osteoartrite/genética , Coelhos , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/fisiologia , Sialoglicoproteínas/genética , Sialoglicoproteínas/fisiologia , Líquido Sinovial/metabolismo , Membrana Sinovial/citologia , Membrana Sinovial/metabolismo , Transfecção/métodosRESUMO
Chitosan-pEGFP nanoparticles were synthesized through the complex coacervation of the cationic polymer with pEGFP, in order to examine the potential of chitosan as a non-viral gene delivery vector to transfer exogenous gene into primary chondrocytes for the treatment of joint diseases. The nanoparticles were prepared at an N/P ratio of 3.8 and showed a spherical or irregular shape. The mean particle size and zeta potential of the nanoparticles freshly prepared with chitosan of different molecular weight were in the range of 100-300 nm and varied from +1 to +23 mV, respectively. Both the particle size and the zeta potential altered in DMEM of different pH. The transfection of primary chondrocytes was performed in different conditions by varying pH of transfection medium, molecular weight of chitosan and different plasmid dosage. Analysis of FACS demonstrated that the transfection efficiency could reach a much high level and the percentage of positive cells could exceed 50% in certain condition. These results suggest that chitosan-DNA nanoparticles have favorable characteristics for non-viral gene delivery to primary chondrocytes, and have the potential to deliver therapeutic genes directly into joint.
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Quitosana/administração & dosagem , Condrócitos/metabolismo , DNA/administração & dosagem , Proteínas de Fluorescência Verde/administração & dosagem , Nanoestruturas , Transfecção/métodos , Animais , Quitosana/química , DNA/química , DNA/genética , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Articulação do Joelho/citologia , Articulação do Joelho/metabolismo , Peso Molecular , CoelhosRESUMO
OBJECTIVE: To identify the androgen-responsive genes in prostate and screen the molecular targets for further studying human prostate cancer. METHODS: The potential androgen-responsive gene pituitary tumor transforming gene 1 (PTTG1) was selected which had been previously screened by cDNA microarray in rat prostate and its mRNA level was detected by Northern blot in the castrated rat prostate with and without replacement of Mibolerone. Immunohistochemistry was performed to determine the expression and location of PTTG1 in human prostate tissues. Then human androgen-dependent prostate cancer cells LNCaP were used as a model to study the regulation of PTTG1 by Mibolerone. RESULTS: PTTG1 mRNA was hardly detectable in the prostate of 7-day castrated rats, while it was up-regulated dramatically in the prostate of 7-day castrated rats treated with Mibolerone for 2 days. It was showed that high expression of PTTG1 was localized to the epithelial cells of human prostate cancer but not to the stromal cells with Immunohistochemistry. Northern blot analysis indicated that LNCaP cells treated with 0.1 nmol/L Mibolerone for 2 days led to the high PTTG1 mRNA expression. The basic expression of PTTG1 in human androgen-independent prostate cancer cell lines PC3 or DU145 was even higher than that in the human androgen-dependent prostate cancer cells LNCaP treated with Mibolerone. CONCLUSION: Androgen can up-regulate the PTTG1 expression in castrated rat prostate and human prostate cancer cell LNCaP. It suggests that PTTG1 is potential to play an important role in human prostate cancer progression.
Assuntos
Expressão Gênica/efeitos dos fármacos , Nandrolona/análogos & derivados , Proteínas de Neoplasias/genética , Próstata/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Northern Blotting , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Masculino , Nandrolona/farmacologia , Proteínas de Neoplasias/metabolismo , Orquiectomia , Próstata/efeitos dos fármacos , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Securina , Congêneres da Testosterona/farmacologiaRESUMO
Insulin-like growth factor binding protein-3 (IGFBP-3) is a well documented growth inhibitor and pro-apoptotic factor. IGFBP-3 mRNA and its protein are overexpressed by senescent human diploid fibroblasts. However, the mechanism responsible for the up-regulation of its expression is still unclear. This report describes a novel transcriptional regulatory element, IGFBP-3 enhancer element (IEE), identified in the 5' untranslated region of the IGFBP-3 gene. This element differentially activates IGFBP-3 expression in senescent versus young fibroblasts. Electrophoretic mobility shift assays revealed abundant complexes in senescent cell nuclear extracts compared with young cell nuclear extracts. Similar to young proliferative cells, young quiescent cells showed reduced binding activity; enhancement of this activity was specific to senescent cells and not an effect of cell cycle arrest. The DNase I footprint revealed the protein-binding core sequence within the IEE through which the protein binds the IEE. Site-directed mutagenesis within IEE abolished binding activity and selectively decreased IGFBP-3 promoter activity in senescent (but not young) cells. Furthermore, introduction of an IEE decoy suppressed the endogenous IGFBP-3 gene expression specifically in senescent cells. These results point to the IEE as being a positive transcription regulatory element that contributes to the up-regulation of IGFBP-3 during cellular senescence.