RESUMO
Ulcerative colitis (UC) is an inflammatory bowel disease with characteristic inflammation to mucosal cells in rectum and colon leading to lesions in mucosa and submucosa. Moreover, crocin is a carotenoid compound among active constituents of saffron with many pharmacological effects as antioxidant, anti-inflammatory and anticancer activities. Therefore, we aimed to investigate therapeutic effects of crocin against UC through affecting the inflammatory and apoptotic pathways. For induction of UC in rats, intracolonic 2 ml of 4% acetic acid was used. After induction of UC, part of rats was treated with 20 mg/kg crocin. cAMP was measured using ELISA. Moreover, we measured gene and protein expression of B-cell lymphoma 2 (BCL2), BCL2-associated X (BAX), caspase-3/8/9, NF-κB, tumor necrosis factor (TNF)-α and IL-1ß/4/6/10. Colon sections were stained with hematoxylin-eosin and Alcian blue or immune-stained with anti-TNF-α antibodies. Microscopic images of colon sections in UC group revealed destruction of intestinal glands associated with infiltration of inflammatory cell and severe hemorrhage. While images stained with Alcian blue showed damaged and almost absent intestinal glands. Crocin treatment ameliorated morphological changes. Finally, crocin significantly reduced expression levels of BAX, caspase-3/8/9, NF-κB, TNF-α, IL-1ß and IL-6, associated with increased levels of cAMP and expression of BCL2, IL-4 and IL-10. In conclusion, protective of action of crocin in UC is proved by restoration of normal weight and length of colon as well as improvement of morphological structure of colon cells. The mechanism of action of crocin in UC is indicated by activation of anti-apoptotic and anti-inflammatory effects.
Assuntos
Colite Ulcerativa , Ratos , Animais , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/tratamento farmacológico , NF-kappa B/metabolismo , Azul Alciano/farmacologia , Caspase 3 , Inibidores do Fator de Necrose Tumoral/uso terapêutico , Proteína X Associada a bcl-2 , Inflamação/tratamento farmacológico , Inflamação/patologia , Carotenoides/farmacologia , Carotenoides/uso terapêutico , Colo/patologia , Fator de Necrose Tumoral alfa/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Apoptose , Modelos Animais de DoençasRESUMO
The molecular mechanism of mechanical force regulating the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) has not been clearly elucidated. In this study, two mRNA-seqs, GSE106887 and GSE109167, which contained several samples of PDLSCs under mechanical force, were downloaded from Gene Expression Omnibus. Differential expression analysis was firstly taken between GSE106887 and GSE109167, then the common 84 up-regulated genes and 26 down-regulated genes were selected. Function enrichment analysis was used to identify the key genes and pathways in PDLSCs subjected to the tension and compression force. PDLSCs were isolated from human periodontal ligament tissues. The effects of ANGPTL4 knockdown with shRNA on the osteogenic differentiation of PDLSCs were studied in vitro. Then, the orthodontic tooth movement (OTM) rat model was used to study the expression of HIF-1α and ANGPTL4 in alveolar bone remodeling in vivo. ANGPTL4 and the HIF-1 pathway were identified in PDLSCs subjected to the tension and compression force. alizarin red staining, alcian blue staining, and oil red O staining verified that PDLSCs had the ability of osteogenic, chondrogenic, and adipogenic differentiation, respectively. Verification experiment revealed that the expression of ANGPTL4 in PDLSCs significantly increased when cultured under osteogenic medium in vitro. While ANGPTL4 was knocked down by shRNA, the levels of ALPL, RUNX2, and OCN decreased significantly, as well as the protein levels of COL1A1, ALPL, RUNX2, and OCN. During the OTM, the expression of HIF-1α and ANGPTL4 in periodontal ligament cells increased on the tension and compression sides. We concluded the positive relationship between ANGPTL4 and osteogenic differentiation of PDLSCs.
Assuntos
Osteogênese , Ligamento Periodontal , Azul Alciano/metabolismo , Azul Alciano/farmacologia , Proteína 4 Semelhante a Angiopoietina/genética , Proteína 4 Semelhante a Angiopoietina/metabolismo , Animais , Diferenciação Celular/genética , Proliferação de Células , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/farmacologia , Humanos , Osteogênese/genética , Ligamento Periodontal/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Células-Tronco/metabolismoRESUMO
This study aimed to compare and analyze the effect of N-cadherin on chondrogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) and to explore the related mechanism, so as to provide a novel theoretical basis for the clinical work of articular cartilage injury regeneration and repair. For this purpose, the experimental animals were clean grade SD rats (aged 5-6 weeks, weighing 180-250g). Alcian blue staining was carried out to observe the induced chondrogenesis following N-cadherin inhibition. The specific role of N-cadherin in the Wnt signaling pathway and chondrogenic differentiation of BMSCs was detected by Western blot; while the effect of N-cadherin on the molecular level changes of ß-catenin in the cytoplasm was evaluated by fluorescence quantitative real-time PCR (qRT-PCR). In addition, immunoprecipitation (IP) was used for the verification of the interaction between N-cadherin and ß-catenin. Results showed that under the light microscope, 90% of the BMSCs at the third generation, 90% of the cells were fused. Alcian blue staining showed that the green staining area in the BMP2 induction group was large and dense, while that in the N-cadherin inhibition group and blank control group was small and sparse. Western blot revealed that N-cadherin and SOX9 were significantly developed in the BMP2 induction group, but Wnt3a was not significantly developed. While in the N-cadherin inhibition group, the development of Wnt3a was obvious, yet without evident development of N-cadherin and SOX9. The qRT-PCR indicated that the relative mRNA expression of Wnt3a was significantly increased in the N-cadherin inhibition group (P<0.05). However, no obvious difference was observed in the mRNA expression of ß-catenin between the BMP2 induction group and the N-cadherin inhibition group (P>0.05). Western blot indicated that in the BMP2 induction group; there existed the development of ß-catenin, significant development of phos-GSK-3ß and total GSK-3ß, but no obvious development of Wnt3a. In the N-cadherin inhibition group, there was significantly enhanced development of Wnt3a and ß-catenin than that before, blurred development of phos-GSK-3ß than that before, and also obvious development of total GSK-3ß with little change from before. N-cadherin promoted the expression of ß-catenin mostly in the cell membrane, but only a few in the cytoplasm and nucleus. Additionally, verification by IP showed that N-cadherin and ß-catenin were developed on N-cadherin and ß-catenin bands, suggesting an interaction between N-cadherin and ß-catenin. According to these results, N-cadherin can ultimately promote chondrogenic differentiation of BMSCs by inhibiting the Wnt signaling pathway.
Assuntos
Condrogênese , Células-Tronco Mesenquimais , Azul Alciano/metabolismo , Azul Alciano/farmacologia , Animais , Medula Óssea , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Condrogênese/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismoRESUMO
OBJECTIVE: This study investigated the molecular mechanism of whether hUC-MSCs-EVs repressed PTEN expression and activated the PI3K/AKT pathway through miR-29b-3p, thus inhibiting LPS-induced neuronal injury. METHODS: hUC-MSCs were cultured and then identified. Cell morphology was observed. Alizarin red, oil red O, and alcian blue staining were used for inducing osteogenesis, adipogenesis, and chondrogenesis. EVs were extracted from hUC-MSCs and identified by transmission electron microscope observation and Western blot. SCI neuron model was established by 24h lipopolysaccharide (LPS) induction. After the cells were cultured with EVs without any treatment, uptake of EVs by SCI neurons, miR-29b-3p expression, cell viability, apoptosis, caspase-3, cleaved caspase-3, caspase 9, Bcl-2, PTEN, PI3K, AKT, and p-Akt protein levels, caspase 3 and caspase 9 activities, and inflammatory factors IL-6 and IL-1ß levels were detected by immunofluorescence labeling, RT-qPCR, MTT, flow cytometry, Western blot, caspase 3 and caspase 9 activity detection kits, and ELISA. The binding sites between PTEN and miR-29b-3p were predicted by the database and verified by dual-luciferase assay. RESULTS: LPS-induced SCI cell model was successfully established, and hUC-MSCs-EVs inhibited LPS-induced apoptosis of injured spinal cord neurons. EVs transferred miR-29b-3p into LPS-induced injured neurons. miR-29b-3p silencing reversed EV effects on reducing LPS-induced neuronal apoptosis. miR-29b-3p reduced LPS-induced neuronal apoptosis by targeting PTEN. After EVs-miR-inhi and si-PTEN treatment, inhibition of the PI3K/AKT pathway reversed hUC-MSCs-EVs effects on reducing LPS-induced neuronal apoptosis. CONCLUSION: hUC-MSCs-EVs activated the PI3K/AKT pathway by carrying miR-29b-3p into SCI neurons and silencing PTEN, thus reducing neuronal apoptosis.
Assuntos
Vesículas Extracelulares , Células-Tronco Mesenquimais , MicroRNAs , Traumatismos da Medula Espinal , Azul Alciano/metabolismo , Azul Alciano/farmacologia , Apoptose , Caspase 3/metabolismo , Caspase 9/metabolismo , Caspase 9/farmacologia , Vesículas Extracelulares/metabolismo , Humanos , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , MicroRNAs/genética , MicroRNAs/metabolismo , Neurônios/metabolismo , PTEN Fosfo-Hidrolase , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/terapia , Cordão Umbilical/metabolismoRESUMO
Mast cells are large cells with granular cytoplasm that participate in wound healing, angiogenesis and defense against pathogens. They also contribute to inflammation by initiating innate and acquired immunity. The granules of these cells exhibit characteristic staining properties. We investigated toluidine blue, astra blue, Alcian blue-pyronin Y and May-Grunwald Giemsa stains for mast cells in various oral lesions and assessed the efficacy of each for identifying mast cells. Sections were obtained from 10 each of diagnosed cases of inflammatory fibrous hyperplasia, periapical cyst, mild dysplasia, oral submucous fibrosis and oral squamous cell carcinoma and stained using the stains listed above. Mast cells were assessed for their presence, contrast of the mast cell in the connective tissue background and number. We found that May-Grunwald Giemsa stain was the best for identification of mast cells, although toluidine blue staining is less time-consuming. Overall we obtained better results using May-Grunwald Giemsa and toluidine blue for staining mast cells.
Assuntos
Carcinoma de Células Escamosas/patologia , Mastócitos/patologia , Neoplasias Bucais/patologia , Azul Alciano/farmacologia , Contagem de Células/métodos , Corantes/farmacologia , Técnicas Histológicas/métodos , Humanos , Coloração e Rotulagem/métodos , Cloreto de Tolônio/farmacologiaRESUMO
Aluminum (Al) is ubiquitous and toxic to microbes. High Al3+ concentration and low pH are two key factors responsible for Al toxicity, but our present results contradict this idea. Here, an Al-tolerant yeast strain Rhodotorula taiwanensis RS1 was incubated in glucose media containing Al with a continuous pH gradient from pH 3.1-4.2. The cells became more sensitive to Al and accumulated more Al when pH increased. Calculations using an electrostatic model Speciation Gouy Chapman Stern indicated that, the increased Al sensitivity of cells was associated with AlOH2+ and Al(OH) 2+ rather than Al3+. The alcian blue (a positively charged dye) adsorption and zeta potential determination of cell surface indicated that, higher pH than 3.1 increased the negative charge and Al adsorption at the cell surface. Taken together, the enhanced sensitivity of R. taiwanensis RS1 to Al from pH 3.1-4.2 was associated with increased hydroxy-Al and cell-surface negativity.
Assuntos
Hidróxido de Alumínio/química , Alumínio/toxicidade , Membrana Celular/fisiologia , Rhodotorula/crescimento & desenvolvimento , Eletricidade Estática , Azul Alciano/farmacologia , Membrana Celular/metabolismo , Concentração de Íons de Hidrogênio , Rhodotorula/efeitos dos fármacos , Rhodotorula/metabolismoRESUMO
In previously published work, we have described heparin-binding synthetic peptides that preferentially recognize amyloid deposits in a mouse model of reactive systemic (AA) amyloidosis and can be imaged by using positron and single photon emission tomographic imaging. We wanted to extend these findings to the most common form of visceral amyloidosis, namely light chain (AL); however, there are no robust experimental animal models of AL amyloidosis. To further define the binding of the lead peptide, p5, to AL amyloid, we characterized the reactivity in vitro of p5 with in situ and patient-derived AL amyloid extracts which contain both hypersulfated heparan sulfate proteoglycans as well as amyloid fibrils. Histochemical staining demonstrated that the peptide specifically localized with tissue-associated AL amyloid deposits. Although we anticipated that p5 would undergo electrostatic interactions with the amyloid-associated glycosaminoglycans expressing heparin-like side chains, no significant correlation between peptide binding and glycosaminoglycan content within amyloid extracts was observed. In contrast, following heparinase I treatment, although overall binding was reduced, a positive correlation between peptide binding and amyloid fibril content became evident. This interaction was further confirmed using synthetic light chain fibrils that contain no carbohydrates. These data suggest that p5 can bind to both the sulfated glycosaminoglycans and protein fibril components of AL amyloid. Understanding these complex electrostatic interactions will aid in the optimization of synthetic peptides for use as amyloid imaging agents and potentially as therapeutics for the treatment of amyloid diseases.
Assuntos
Amiloide/metabolismo , Amiloidose/metabolismo , Glicosaminoglicanos/química , Heparina Liase/química , Peptídeos/farmacologia , Azul Alciano/química , Azul Alciano/farmacologia , Benzotiazóis , Carboidratos/química , Glicosaminoglicanos/metabolismo , Heparina/química , Humanos , Peptídeos/química , Ligação Proteica , Eletricidade Estática , Tiazóis/farmacologiaRESUMO
The cartilaginous elements forming the pharyngeal arches of the zebrafish derive from cranial neural crest cells. Their proper differentiation and patterning are regulated by reciprocal interactions between neural crest cells and surrounding endodermal, ectodermal and mesodermal tissues. In this study, we show that the endodermal factors Runx3 and Sox9b form a regulatory cascade with Egr1 resulting in transcriptional repression of the fsta gene, encoding a BMP antagonist, in pharyngeal endoderm. Using a transgenic line expressing a dominant negative BMP receptor or a specific BMP inhibitor (dorsomorphin), we show that BMP signaling is indeed required around 30 hpf in the neural crest cells to allow cell differentiation and proper pharyngeal cartilage formation. Runx3, Egr1, Sox9b and BMP signaling are required for expression of runx2b, one of the key regulator of cranial cartilage maturation and bone formation. Finally, we show that egr1 depletion leads to increased expression of fsta and inhibition of BMP signaling in the pharyngeal region. In conclusion, we show that the successive induction of the transcription factors Runx3, Egr1 and Sox9b constitutes a regulatory cascade that controls expression of Follistatin A in pharyngeal endoderm, the latter modulating BMP signaling in developing cranial cartilage in zebrafish.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição SOX9/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Azul Alciano/farmacologia , Animais , Cartilagem/metabolismo , Diferenciação Celular , Endoderma/metabolismo , Epitélio/metabolismo , Feminino , Folistatina/metabolismo , Masculino , Crista Neural/citologia , Oligonucleotídeos/metabolismo , Pirazóis/metabolismo , Pirimidinas/metabolismo , Transdução de Sinais , Crânio/embriologia , Crânio/metabolismo , Fatores de Tempo , Peixe-ZebraRESUMO
Restrictive adhesions are a common complication of tendon injury and repair in the hand, resulting in severe dysfunction. Creating a barrier between the repair sites and surrounding tissue layers may prevent adhesions. We present the first stage in the process of developing a synovial biomembrane for this purpose. Synovial cells harvested from the Achilles tendon sheath and the knee joint of a Wistar albino rat were cultured for 2 weeks in culture medium, and then impregnated into a collagen type 1 matrix for another 2 weeks. Cells originating from both tendon and synovium demonstrated cell growth and layer formation on the surfaces of the matrix 2 weeks after impregnation. Alcian blue staining using Scott's method demonstrated the presence of acidic mucopolysaccharide, indicating hyaluronic acid (HA) production. This provides indirect evidence of functioning synovial cells on the membrane. It is possible to culture synovial cells and engineer a synoviocyte-collagen membrane that synthesizes endogenous HA. Application of this biomembrane to tendon repair sites may help to prevent adhesions after tendon repairs. Evaluation of this method on in vivo models is required.
Assuntos
Materiais Biocompatíveis/química , Ácido Hialurônico/metabolismo , Membrana Sinovial/citologia , Tendões/patologia , Engenharia Tecidual/métodos , Azul Alciano/farmacologia , Animais , Células Cultivadas , Corantes/farmacologia , Células do Tecido Conjuntivo/citologia , Ratos , Líquido Sinovial/citologia , Traumatismos dos Tendões/cirurgia , Aderências Teciduais , CicatrizaçãoRESUMO
Chondrogenesis is a critical step in palatogenesis. All-trans retinoic acid (atRA), a vitamin A derivative, is a known teratogenic effector of cleft palate. Here, we evaluated the effects of atRA on the osteo-/chondrogenic differentiation of mouse embryonic palate mesenchymal (MEPM) cells. MEPM cells, in a high-density micromass environment, undergo active chondrogenesis in a manner analogous to that of limb-derived mesenchymal cells, and served as a valid model system to investigate the mechanisms regulating chondrogenesis during palatogenesis. atRA-treated MEPM micromass expressed relatively higher levels of osteoblastic gene markers (alkaline phosphatase and collagen type I) and lower levels of chondrocytic gene markers (collagen type II and aggrecan). As transforming growth factor-beta3 (TGF-beta3) is an essential growth factor for chondrogenesis of embryonic mesenchymal cells both in in vivo and in vitro conditions, we thereby explored the effects of atRA on TGF-beta3 signaling pathway. atRA led to an increase in mRNA expression of TGF-beta3 and an instantaneous decrease in TGF-beta type II receptor (TbetaRII) as determined by real-time RT-PCR. Further study showed that atRA inhibited phosphorylation of Smad2 and Smad3 and increased Smad7 expression. Activation of the Smad pathways by transfection with Smad7deltaC mutant or constitutively active TbetaRII retroviral vector abolished atRA-induced inhibition of chondrogenesis as indicated by Alcian blue staining, indicating that Smad signaling is essential for this response. Taken together, these data for the first time demonstrated a role for RA-induced hypochondrogenesis through regulation of the TGF-beta3 pathway and suggested a role for TbetaRII /Smad in retinoid-induced cleft palate.
Assuntos
Condrócitos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Palato/embriologia , Proteínas Smad/metabolismo , Tretinoína/metabolismo , Azul Alciano/metabolismo , Azul Alciano/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Western Blotting , Linhagem da Célula , Células Cultivadas , Colágeno Tipo I/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Camundongos , Mutação , Osteoblastos/metabolismo , Osteogênese , Fenótipo , Fosforilação , Proteínas Serina-Treonina Quinases , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Proteína Smad7/metabolismo , Fatores de Tempo , Transfecção , Fator de Crescimento Transformador beta/metabolismoRESUMO
UNLABELLED: Runx proteins mediate skeletal development. We studied the regulation of Runx1 during chondrocyte differentiation by real-time RT-PCR and its function during chondrogenesis using overexpression and RNA interference. Runx1 induces mesenchymal stem cell commitment to the early stages of chondrogenesis. INTRODUCTION: Runx1 and Runx2 are co-expressed in limb bud cell condensations that undergo both cartilage and bone differentiation during murine development. However, the cooperative and/or compensatory effects these factors exert on skeletal formation have yet to be elucidated. MATERIALS AND METHODS: Runx1/Cbfa2 and Runx2/Cbfa1 were examined at different stages of embryonic development by immunohistochemistry. In vitro studies used mouse embryonic limb bud cells and assessed Runx expressions by immunohistochemistry and real-time RT-PCR in the presence and absence of TGFbeta and BMP2. Runx1 was overexpressed in mesenchymal cell progenitors using retroviral infection. RESULTS: Immunohistochemistry showed that Runx1 and Runx2 are co-expressed in undifferentiated mesenchyme, had similar levels in chondrocytes undergoing transition from proliferation to hypertrophy, and that there was primarily Runx2 expression in hypertrophic chondrocytes. Overall, the expression of Runx1 remained significantly higher than Runx2 mRNA levels during early limb bud cell maturation. Treatment of limb bud micromass cultures with BMP2 resulted in early induction of both Runx1 and Runx2. However, upregulation of Runx2 by BMP2 was sustained, whereas Runx1 decreased in later time-points when type X collagen was induced. Although TGFbeta potently inhibits Runx2 and type X collagen, it induces type II collagen mRNA and mildly but significantly inhibits Runx1 isoforms in the early stages of chondrogenesis. Virus-mediated overexpression of Runx1 in mouse embryonic mesenchymal cells resulted in a potent induction of the early chondrocyte differentiation markers but not the hypertrophy marker, type X collagen. Knockdown or Runx1 potently inhibits type II collagen, alkaline phosphatase, and Runx2 and has a late inhibitory effect on type X collagen. CONCLUSION: These findings show a distinct and sustained role for Runx proteins in chondrogenesis and subsequent chondrocyte maturation. Runx1 is highly expressed during chondrogenesis in comparison with Runx2, and Runx1 gain of functions stimulated this process. Thus, the Runx genes are uniquely expressed and have distinct roles during skeletal development.
Assuntos
Diferenciação Celular , Condrócitos/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Mesoderma/metabolismo , Azul Alciano/farmacologia , Animais , Western Blotting , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/metabolismo , Osso e Ossos , Cartilagem/citologia , Cartilagem/metabolismo , Proliferação de Células , Colágeno Tipo II/metabolismo , Colágeno Tipo X/metabolismo , Corantes/farmacologia , Primers do DNA/química , Imuno-Histoquímica , Hibridização In Situ , Óperon Lac , Botões de Extremidades/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos ICR , Fenótipo , Isoformas de Proteínas , RNA/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/citologia , Fatores de Tempo , Fator de Crescimento Transformador beta/metabolismo , Regulação para CimaRESUMO
The reconstruction of soft tissue defects remains a challenge in plastic and reconstructive surgery, and a real clinical need exists for an adequate solution. This study was undertaken in order to differentiate mesenchymal stem cells (MSCs) into adipocytes, and to then assess the possibility of constructing adipose tissue via the attachment of MSCs to injectable PLGA spheres. We also designed injectable PLGA spheres for scar-free transplantation. In this study, MSCs and adipo-MSCs (MSCs cultured in adipogenic medium for 7 days) were attached to PLGA spheres and cultured for 7 days, followed by injection into nude mice for 2 weeks. As a result, the difference between lipid accumulation in adipo-MSCs at 1 and 7 days was much higher in vitro than in the MSCs. Two weeks after injection, a massive amount of new tissue was formed in the APLGA group, whereas only a small amount was formed in the MPLGA group. We verified that the newly formed tissue originated from the injected MSCs via GFP testing, and confirmed that the created tissue was actual adipose tissue by oil red O staining and Western blot (PPAR(gamma) and C/EBP(alpha) were expressed only in APLGA groups). Therefore, this study presents an efficient model of adipose tissue engineering using MSCs and injectable PLGA spheres.
Assuntos
Tecido Adiposo/patologia , Ácido Láctico/química , Células-Tronco Mesenquimais/citologia , Microesferas , Ácido Poliglicólico/química , Polímeros/química , Engenharia Tecidual/métodos , Adipócitos/citologia , Azul Alciano/farmacologia , Animais , Antraquinonas/farmacologia , Compostos Azo/farmacologia , Western Blotting , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Diferenciação Celular , Proliferação de Células , Transplante de Células , Cicatriz , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Hexosaminidases/metabolismo , Metabolismo dos Lipídeos , Camundongos , Camundongos Nus , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Coelhos , Fatores de Tempo , TransfecçãoRESUMO
Biomechanical forces are major epigenetic factors that determine the form and differentiation of skeletal tissues, and may be transduced through cell adhesion to the intracellular biochemical signaling pathway. To test the hypothesis that stepwise stretching is translated to molecular signals during early chondrogenesis, we developed a culture system to study the proliferation and differentiation of chondrocytes. Rat embryonic day-12 limb buds were microdissected and dissociated into cells, which were then micromass cultured on a silicone membrane and maintained for up to 7 days. Stepwise-increased stretching was applied to the silicone membrane, which exerted shearing stress on the cultures on day 4 after the initiation of chondrogenesis. Under stretched conditions, type II collagen expression was significantly inhibited by 44% on day 1 and by 67% on day 2, and this difference in type II collagen reached 80% after 3 days of culture. Accumulation of type II collagen protein and the size of the chondrogenic nodules had decreased by 50% on day 3. On the other hand, expression of the non-chondrogenic marker fibronectin was significantly upregulated by 1.8-fold on day 3, while the up-regulation of type I collagen was minimal, even by day 3. The downregulation in the expression of chondrogenic markers was completely recovered when cell-extracellular matrix attachment was inhibited by Gly-Arg-Gly-Asp-Ser-Pro-Lys peptide or by the application of blocking antibodies for alpha2, alpha5 or beta1 integrins. We conclude that shearing stress generated by stepwise stretching inhibits chondrogenesis through integrins, and propose that signal transduction from biomechanical stimuli may be mediated by cell-extracellular matrix adhesion.
Assuntos
Condrócitos/citologia , Condrogênese , Botões de Extremidades/embriologia , Azul Alciano/farmacologia , Animais , Fenômenos Biomecânicos , Western Blotting , Adesão Celular , Proliferação de Células , Junções Célula-Matriz , Condrócitos/metabolismo , Colágeno/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo II/metabolismo , Técnicas de Cultura , Fibronectinas/metabolismo , Integrinas/metabolismo , Mesoderma/metabolismo , Microscopia de Fluorescência , Oligopeptídeos/farmacologia , Peptídeos/química , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Silício/metabolismo , Fatores de Tempo , Regulação para CimaRESUMO
The cell adhesion molecule N-cadherin is implicated in many morphogenetic processes, including mesenchyme condensation during limb development. To further understand N-cadherin function, we characterized a new N-cadherin allele containing the lacZ reporter gene under the regulation of the mouse N-cadherin promoter. The reporter gene recapitulates the expression pattern of the N-cadherin gene, including expression in heart, neural tube, and somites. In addition, strong expression was observed in areas of active cellular condensation, a prerequisite for chondrogenic differentiation, including the developing mandible, vertebrae, and limbs. Previous studies from our laboratory have shown that limb buds can form in N-cadherin-null embryos expressing a cardiac-specific cadherin transgene, however, these partially rescued embryos do not survive long enough to observe limb development. To overcome the embryonic lethality, we used an organ culture system to examine limb development ex vivo. We demonstrate that N-cadherin-deficient limb buds were capable of mesenchymal condensation and chondrogenesis, resulting in skeletal structures. In contrast to previous studies in chicken using N-cadherin-perturbing antibodies, our organ culture studies with mouse tissue demonstrate that N-cadherin is not essential for limb mesenchymal chondrogenesis. We postulate that another cell adhesion molecule, possibly cadherin-11, is responsible for chondrogenesis in the N-cadherin-deficient limb.
Assuntos
Caderinas/fisiologia , Extremidades/embriologia , Mesoderma/metabolismo , Azul Alciano/metabolismo , Azul Alciano/farmacologia , Alelos , Animais , Caderinas/metabolismo , Adesão Celular , Condrogênese , Cruzamentos Genéticos , Técnica Indireta de Fluorescência para Anticorpo , Galactosídeos , Genes Reporter , Técnicas Genéticas , Heterozigoto , Homozigoto , Indóis , Óperon Lac , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Modelos Genéticos , Técnicas de Cultura de Órgãos , Regiões Promotoras Genéticas , Retroviridae/genética , Sequências Repetidas Terminais , Fatores de Tempo , Transgenes , beta-Galactosidase/metabolismoRESUMO
The synthesis and contents of extracellular non-collagenous matrix macromolecules was studied in early and late human osteoarthritic (OA) cartilage obtained at surgery for sarcomas in the lower extremities (normal and early OA) or for total knee replacement (late stage OA). The early OA samples were those that had some fibrillation in the joint by visual examination. One group had fibrillation in the area sampled and the other group had no fibrillation. Cartilage was taken from the same topographical area on the medial femoral condyle in all the samples, labeled with [3H]leucine and [35S]sulfate for 4 h at 37 degrees C and extracted with 4 M guanidine-HCl. Analysis of the extracts showed that the total amount of proteoglycans relative to hydroxyproline content was higher in the early and late OA than in the normal cartilage. These proteoglycans showed a relatively lower [35S]sulfate incorporation into GAG chains and a higher [3H]leucine incorporation. The pattern of newly synthesized proteins was altered similarly in early and late OA. Notably, synthesis of cartilage oligomeric matrix protein (COMP), fibronectin, and cartilage intermediate layer protein (CILP) was increased, also reflected in their abundance as determined by enzyme-linked immunosorbent assay (ELISA). Collagen synthesis appeared significantly increased only in the late stage OA. The observed altered composition and pattern of biosynthesis indicate that the joint undergoes metabolic alterations early in the disease process, even before there is overt fibrillation of the tissue. The early OA samples studied appear to represent two distinct groups of early lesions in different stages of the process of cartilage deterioration as shown by their differences in relative rates of synthesis and abundance of proteins.
Assuntos
Matriz Extracelular/metabolismo , Osteoartrite/metabolismo , Idoso , Azul Alciano/farmacologia , Peso Corporal , Cartilagem/metabolismo , Cartilagem Articular/metabolismo , Cromatografia , Cromatografia por Troca Iônica , Colágeno/química , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Hidroxiprolina/química , Leucina/química , Masculino , Pessoa de Meia-Idade , Osteoartrite/patologia , Pró-Colágeno/química , Proteoglicanas/química , Proteoglicanas/metabolismo , TemperaturaRESUMO
Mutations in the human cartilage oligomeric matrix protein (COMP) gene have been linked to the development of pseudoachondroplasia and multiple epiphyseal dysplasia. We previously cloned the promoter region of the COMP gene and delineated a minimal negative regulatory element (NRE) that is both necessary and sufficient to repress its promoter (Issack, P. S., Fang, C. H., Leslie, M. P., and Di Cesare, P. E. (2000) J. Orthop. Res. 18, 345-350; Issack, P. S., Liu, C. J., Prazak, L., and Di Cesare, P. E. (2004) J. Orthop. Res. 22, 751-758). In this study, a yeast one-hybrid screen for proteins that associate with the NRE led to the identification of the leukemia/lymphoma-related factor (LRF), a transcriptional repressor that contains a POZ (poxvirus zinc finger) domain, as an NRE-binding protein. LRF bound directly to the NRE both in vitro and in living cells. Nine nucleotides (GAGGGTCCC) in the 30-bp NRE are essential for binding to LRF. LRF showed dose-dependent inhibition of COMP-specific reporter gene activity, and exogenous overexpression of LRF repressed COMP gene expression in both rat chondrosarcoma cells and bone morphogenetic protein-2-treated C3H10T1/2 progenitor cells. In addition, LRF also inhibited bone morphogenetic protein-2-induced chondrogenesis in high density micromass cultures of C3H10T1/2 cells, as evidenced by lack of expression of other chondrocytic markers, such as aggrecan and collagen types II, IX, X, and XI, and by Alcian blue staining. LRF associated with histone deacetylase-1 (HDAC1), and experiments utilizing the HDAC inhibitor trichostatin A revealed that LRF-mediated repression requires deacetylase activity. LRF is the first transcription factor found to bind directly to the COMP gene promoter, to recruit HDAC1, and to regulate both COMP gene expression and chondrogenic differentiation.
Assuntos
Cartilagem/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Azul Alciano/farmacologia , Motivos de Aminoácidos , Animais , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Condrossarcoma/embriologia , Condrossarcoma/metabolismo , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Clonagem Molecular , Relação Dose-Resposta a Droga , Genes Reporter , Glutationa Transferase/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Immunoblotting , Imunoprecipitação , Magnetismo , Camundongos , Camundongos Endogâmicos C3H , Dados de Sequência Molecular , Mutação , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Transcrição Gênica , Técnicas do Sistema de Duplo-Híbrido , Dedos de ZincoRESUMO
Colon cancers develop after accumulation of multiple genetic and epigenetic alterations in colon epithelial cells. To shed light on global changes in gene expression of colon cancers and to gain further insight into the molecular mechanisms underlying colon carcinogenesis, we have conducted a comprehensive microarray analysis of mRNA using a rat colon cancer model with the food-borne carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Of 8749 genes or ESTs on a high density oligonucleotide microarray, 27 and 46 were over- and underexpressed, respectively, by > or =3-fold in colon cancers in common in two rat strains with distinct susceptibility to PhIP carcinogenesis. For example, genes involved in inflammation and matrix proteases and a cell cycle regulator gene, cyclin D2, were highly expressed in colon cancers. In contrast, genes encoding structural proteins, muscle-related proteins, matrix-composing and mucin-like proteins were underexpressed. Interestingly, a subset of genes whose expression is characteristic of Paneth cells, i.e. the defensins and matrilysin, were highly overexpressed in colon cancers. The presence of defensin 3 and defensin 5 transcripts in cancer cells could also be confirmed by in situ mRNA hybridization. Furthermore, Alcian blue/periodic acid Schiff base (AB-PAS) staining and immunohistochemical analysis with an anti-lysozyme antibody demonstrated Paneth cells in the cancer tissues. AB-PAS-positive cells were also observed in high grade dysplastic aberrant crypt foci, which are considered to be preneoplastic lesions of the colon. Our results suggest that Paneth cell differentiation in colon epithelial cells could be an early morphological change in cryptic cells during colon carcinogenesis.
Assuntos
Carcinógenos , Neoplasias do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica , Imidazóis , Azul Alciano/farmacologia , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Neoplasias do Colo/induzido quimicamente , Ciclina D2 , Ciclinas/metabolismo , Defensinas/metabolismo , Etiquetas de Sequências Expressas , Humanos , Imuno-Histoquímica , Muramidase/química , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos/química , Celulas de Paneth/citologia , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos F344 , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The urokinase-type plasminogen activator receptor (u-PAR) plays a central role in cell migration, growth, and invasion and is regulated, in part, transcriptionally. In mice, u-PAR expression is restricted to a few tissues, one of which is the colon. We therefore screened a colon expression library for regulators of u-PAR promoter activity and identified a zinc finger protein bearing consensus sequences to the Kruppel-like family of transcription factors and showing partial homology with one of the members, KLF4. Like u-PAR, KLF4 expression is predominant in the luminal surface epithelial cells of the colonic crypt, and we hypothesized that u-PAR synthesis in these cells is directed by this transcription factor. Colon cells from KLF4 null mice showed a dramatic reduction in u-PAR protein compared with wild-type mice. Conversely, KLF4 expression in HCT116 colon cancer cells increased the amount of u-PAR protein/mRNA. Transient transfection of KLF4 with a reporter driven by 5'-deleted u-PAR promoter fragments indicated the requirement of the proximal 200 base pairs for optimal expression. Mobility-shifting experiments demonstrated binding of KLF4 to multiple regions of the u-PAR promoter (-154/-128, -105/-71, and -51/-24), and chromatin immunoprecipitation assays confirmed the binding of KLF4 to the endogenous promoter. Deletion of the -144/-123 promoter region diminished but did not eliminate the ability of KLF4 to transactivate the u-PAR promoter, suggesting cooperativity of these binding sites with respect to activation of gene expression. In conclusion, we have identified KLF4 as a novel regulator of u-PAR expression that drives the synthesis of u-PAR in the luminal surface epithelial cells of the colon.
Assuntos
Colo/citologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Células Epiteliais/metabolismo , Receptores de Superfície Celular/biossíntese , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Azul Alciano/farmacologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Northern Blotting , Western Blotting , Linhagem Celular Tumoral , Separação Celular , Cromatina/metabolismo , Clonagem Molecular , Colo/metabolismo , Colo/patologia , DNA Complementar/metabolismo , Epitélio/metabolismo , Citometria de Fluxo , Biblioteca Gênica , Genes Reporter , Proteínas de Fluorescência Verde , Humanos , Imuno-Histoquímica , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Testes de Precipitina , Regiões Promotoras Genéticas , Ligação Proteica , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Transfecção , Regulação para Cima , Dedos de ZincoRESUMO
Fragments of ileum from 663 pigs were collected in abattoirs, prepared with the use of standard histological methods and stained with a novel sensitive histochemical method for the detection of porcine proliferative enteropathy. The method is a combination of the following 3 well-known methods, the Warthin-Starry method, alcian blue and hematoxylin-eosin. In 11 cases, mucus-producing cells were completely absent, severe adenomatous proliferation was observed and intracellular bacteria were found in enterocytes. Disappearance of goblet cells and the presence of adenomatous proliferation without any detectable intracellular bacteria were observed in 16 cases. In the remaining 636 cases, histological changes and intracellular bacteria were not found. When comparing the conventional Warthin-Starry method with the modified staining method presented here, the same 16 cases were found. However, the method presented here enables examination of large numbers of sections in a relatively short period of time.
Assuntos
Azul Alciano/farmacologia , Corantes/farmacologia , Enteropatias/diagnóstico , Mucosa Intestinal/microbiologia , Lawsonia (Bactéria)/isolamento & purificação , Lawsonia (Bactéria)/metabolismo , Coloração pela Prata , Doenças dos Suínos/diagnóstico , Animais , Divisão Celular , Amarelo de Eosina-(YS)/farmacologia , Infecções por Bactérias Gram-Negativas/diagnóstico , Infecções por Bactérias Gram-Negativas/patologia , Hematoxilina/farmacologia , Íleo , Imuno-Histoquímica/métodos , Enteropatias/microbiologia , Enteropatias/patologia , Mucosa Intestinal/patologia , Suínos , Doenças dos Suínos/patologia , Fatores de TempoRESUMO
The copper containing phthalocyanine dyes, alcian blue, copper phthalocyanine tetrasulfonic acid, and Luxol fast blue MBSN are found to induce rapid calcium efflux from actively loaded sarcoplasmic reticulum (SR) vesicles. Alcian blue (5 microM), with 1 mM free Mg2+ triggered Ca2+ efflux at rates greater than 20 nmol/mg of SR/s. As in the case of Ca2+ efflux induced by calcium, heavy metals, or SH oxidation with Cu2+/cysteine, efflux induced by phthalocyanines is also stimulated by adenine containing nucleotides and inhibited by millimolar Mg2+ and submicromolar ruthenium red (RR). In addition, analogs of RR, such as hexamminecobalt(III) chloride or hexammineruthenium(III) chloride also inhibit Ca2+ efflux but are effective at somewhat higher concentrations (approximately 50 microM). Calcium release stimulated by phthalocyanines is specific for SR derived from the terminal cisternae region rather than longitudinal SR. Preincubation of alcian blue with the reducing agents, sodium dithionite, dithiothreitol, or cysteine causes complete loss of Ca2+ release activity from SR vesicles. Reoxidation of the alcian blue leads to return of the Ca2+ release activity of the phthalocyanine dye. The copper containing phthalocyanine dyes appear to cause rapid Ca2+ release from SR vesicles by oxidizing sulfhydryl groups associated with the calcium release channel. Moreover, phthalocyanines appear to act by oxidizing a pair of neighboring sulfhydryls to a disulfide because subsequent additions of the reducing agent dithiothreitol promote the closure of the Ca2+ channel and calcium re-uptake.