RESUMEN
OBJECTIVES: Epulis is considered to be a massive reactive lesion rather than a true neoplasia. AhR is thought to be associated with inflammation and development of neoplasms. Here, we aimed to observe the expression of AhR in fibrous epulis and explore its role and possible mechanism in the pathogenesis of epulis. MATERIALS AND METHODS: Epulis and normal gingival tissues were collected, and AhR expression was detected at the mRNA and protein levels by quantitative polymerase chain reaction (qPCR) and immunohistochemistry, respectively. The expression levels of proinflammatory cytokines and apoptosis-related factor genes in human periodontal ligament cells (hPDLCs) and human gingival fibroblasts (hGFs) transfected with AhR short interfering RNA (siRNA) or negative control siRNA, upon stimulation with lipopolysaccharide of Porphyromonas gingivalis (Pg-LPS), were then examined. Finally, the expression levels of the proinflammatory cytokines and apoptosis-related factor genes in the epulis tissues were observed by qPCR. RESULTS: AhR expression in fibrous epulis was significantly increased at both the mRNA and protein levels. The expression of proinflammatory cytokines and apoptosis-related factor genes in hPDLCs transfected with AhR siRNA was significantly decreased when stimulated with Pg-LPS. The same trends were observed for hGFs. The opposite trend was detected in the epulis tissues. CONCLUSION: AhR may be a key factor in fibrous epulis pathogenesis that acts by regulating the expression of BCL2 family genes and inflammatory factor-related genes.
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Enfermedades de las Encías , Receptores de Hidrocarburo de Aril , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Células Cultivadas , Citocinas/metabolismo , Fibroblastos , Encía/patología , Enfermedades de las Encías/patología , Humanos , Lipopolisacáridos/farmacología , Porphyromonas gingivalis/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , ARN Mensajero , ARN Interferente Pequeño/genética , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Receptores InmunológicosRESUMEN
Bioreducible crosslinked polyplexes from branched polyethylenimine (BPEI, 10â¯kDa) were successfully constructed through DNA neutralization by disulfide-linked azidated BPEI (PAZ) and subsequent DNA condensation by azadibenzocyclooctyne-modified BPEI (PDB), following their self-crosslinking via azide-azadibenzocyclooctyne click chemistry. Click-crosslinked cationic polyplexes (c-polyplexes) revealed high extracellular colloidal stability against negative heparin and ions while intracellular bioreducible degradability for efficient gene unpacking. In vitro gene transfection in cancer cells indicated that the c-polyplexes produced markedly higher transfection efficiency than non-crosslinked counterparts in the serum. The c-polyplexes also had prolonged circulation kinetics, elevated gene accumulation level in SKOV-3 tumor xenografted in a mouse model and in turn superior transgene expression in the tumor. By small hairpin RNA for VEGF silencing, the c-polyplexes exerted significant tumor growth inhibition following with low systemic toxicity in the mouse. This study highlights the design of clickable polycations to construct crosslinked cationic nanopolyplexes for intravenous gene delivery against cancer.
Asunto(s)
Cationes/química , Química Clic/métodos , Terapia Genética/métodos , Polietileneimina/química , Cinética , Factor A de Crecimiento Endotelial Vascular/químicaRESUMEN
Injectable dextran-based hydrogels were prepared for the first time by bioorthogonal click chemistry for cartilage tissue engineering. Click-crosslinked injectable hydrogels based on cyto-compatible dextran (Mw=10kDa) were successfully fabricated under physiological conditions by metal-free alkyne-azide cycloaddition (click) reaction between azadibenzocyclooctyne-modified dextran (Dex-ADIBO) and azide-modified dextran (Dex-N3). Gelation time of these dextran hydrogels could be regulated in the range of approximately 1.1 to 10.2min, depending on the polymer concentrations (5% or 10%) and ADIBO substitution degree (DS, 5 or 10) of Dex-ADIBO. Rheological analysis indicated that the dextran hydrogels were elastic and had storage moduli from 2.1 to 6.0kPa with increasing DS of ADIBO from 5 to 10. The in vitro tests revealed that the dextran hydrogel crosslinked from Dex-ADIBO DS 10 and Dex-N3 DS 10 at a polymer concentration of 10% could support high viability of individual rabbit chondrocytes and the chondrocyte spheroids encapsulated in the hydrogel over 21days. Individual chondrocytes and chondrocyte spheroids in the hydrogel could produce cartilage matrices such as collagen and glycosaminoglycans. However, the chondrocyte spheroids produced a higher content of matrices than individual chondrocytes. This study indicates that metal-free click chemistry is effective to produce injectable dextran hydrogels for cartilage tissue engineering.
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Cartílago/fisiología , Química Clic/métodos , Dextranos/química , Hidrogeles/química , Inyecciones , Ingeniería de Tejidos/métodos , Animales , Supervivencia Celular , Células Cultivadas , Condrocitos/citología , Reactivos de Enlaces Cruzados/química , Dextranos/síntesis química , Metales , Espectroscopía de Protones por Resonancia Magnética , Conejos , Espectroscopía Infrarroja por Transformada de Fourier , Esferoides Celulares/citología , Coloración y EtiquetadoRESUMEN
A novel 4-arm poly(ethylene glycol)-b-poly(disulfide histamine) copolymer was synthesized by Michael addition reaction of poly(ethylene glycol) (PEG) vinyl sulfone and amine-capped poly(disulfide histamine) oligomer, being denoted as 4-arm PEG-SSPHIS. This copolymer was able to condense DNA into nanoscale polyplexes (<200 nm in average diameter) with almost neutral surface charge (+(5-10) mV). Besides, these polyplexes were colloidal stable within 4 h in HEPES buffer saline at pH 7.4 (physiological environment), but rapidly dissociated to liberate DNA in the presence of 10 mM glutathione (intracellular reducing environment). The polyplexes also revealed pH-responsive surface charges which markedly increased with reducing pH values from 7.4-6.3 (tumor microenvironment). In vitro transfection experiments showed that polyplexes of 4-arm PEG-SSPHIS were capable of exerting enhanced transfection efficacy in MCF-7 and HepG2 cancer cells under acidic conditions (pH 6.3-7.0). Moreover, intravenous administration of the polyplexes to nude mice bearing HepG2-tumor yielded high transgene expression largely in tumor rather other normal organs. Importantly, this copolymer and its polyplexes had low cytotoxicity against the cells in vitro and caused no death of the mice. The results of this study indicate that 4-arm PEG-SSPHIS has high potential as a dual responsive gene delivery vector for cancer gene therapy.
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Polietilenglicoles/química , Polímeros/química , Animales , Células COS , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Vectores Genéticos/metabolismo , Células Hep G2 , Humanos , Concentración de Iones de Hidrógeno , Células MCF-7 , Ratones , Ratones Desnudos , Células 3T3 NIH , Oxidación-Reducción , Polímeros/síntesis química , Polímeros/toxicidad , Transfección , Trasplante HeterólogoRESUMEN
OBJECTIVE: To evaluate the survival of bone marrow mesenchymal stem cells (BMMSC) and periodontal ligament stem cells (PDLSC) in BMMSC/PDLSC cell sheets which transplanted ectopically into subcutaneous dorsum of nude mice. METHODS: The canine BMMSC and PDLSC from primary culture were tranfected with lentiviral vectors carrying green fluorescent protein (GFP) gene (Lentivirus-GFP) or red fluorescent protein (RFP) gene (Lentivirus-RFP) respectively. The immunophenotypes of GFP-labeled BMMSC and RFP-labeled PDLSC were identified by flow cytometry. Adipogenic and osteogenic differentiation of them were detected by alizarin red or oil red O respectively. Then, both GFP-labeled BMMSC cell sheets and RFP-labeled PDLSC cell sheets were fabricated respectively using normal culture dish (6 cm) after stimulation of extracellular matrix formation. Each was enveloped by collagen membrane (Bio-Gide) and then transplanted into the subcutaneous dorsum of nude mice. In vivo non-invasive biofluorescence imaging(BFI) was performed at 1, 2, 4 and 8 w post-tranplantation to trace and quantify the survival and growth of RFP-labeled PDLSC and GFP-labeled BMMSC via the BFI system of the NightOWL. The fluorescence intensity change of GFP/RFP signal was monitored and compared. The mice were sacrificed 8 weeks after cell sheets transplantation and the survival of stem cells was verified by fluorescence immunohistochemistry. RESULTS: The flow cytometry showed that GFP-labeled BMMSC positively expressed CD29, CD44, CD34, STRO-1 were 93.07%, 92.84%, 3.23%, 67.67%, and RFP-labeled PDLSCs were 89.91%, 88.47%, 6.04%, 74.11%, respectively. Both of them had the potency of differentiating into osteoblasts and adipocytes. The stemness of both of them was almost same. After being transplanted into nude mice, the signal strength of GFP(BMMSC) was weaker and weaker in 1, 2, 4 and 8 w [(83.1±3.1)×10(6), (65.1±2.3)×10(6), (51.5 ± 2.3)×10(6), (33.8 ± 2.0)×10(6) ph/s, respectively.]. The signal strength of RFP(PDLSC) was weakenedin 1, 2 and 4 w [(53.8±3.0)×10(6), (42.2±2.6)×10(6), (34.5±2.1)×10(6) ph/s], then recovered in 8 w ([ 45.1±2.9)×10(6) ph/s]. The signal strength of RFP(PDLSC) was signifcantly stronger in 8 w than in 4 w(P < 0.01). The survival of RFP-labeled PDLSC was significant higher than that of GFP-labeled BMMSC. After 8 weeks, lots of RFP-labeled PDLSC were observed by microscope, but less GFP-labeled BMMSC were observed. CONCLUSIONS: Histometric analysis revealed that the survival of stem cells in the RFP-labeled PDLSC cell sheets was significantly higher than that of in the GFP-labeled BMMSCs cell sheets.
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Células de la Médula Ósea , Supervivencia Celular , Células Madre Mesenquimatosas , Ligamento Periodontal/citología , Animales , Diferenciación Celular , Colágeno , Perros , Citometría de Flujo , Lentivirus , Proteínas Luminiscentes , Ratones , Ratones Desnudos , Osteoblastos , Osteogénesis , Proteína Fluorescente RojaRESUMEN
The occurrence of three canals in mandibular premolars is very rare. This article reported and discussed the treatment of a mandibular first premolar with 3 root canals, specially in aspect of root-detecting and root-shaping.