RESUMEN
Fibrosis, tightly associated with wound healing, is a significant symptomatic clinical problem. Inflammatory response was reported to be one of the reasons. MiR-155 is relatively related with the development and requirement of inflammatory cells, so we thought reduce the expression of miR-155 in wound sites could improve the quality of healing through reduce inflammatory response. To test this hypothesis, locally antagonizing miR-155 by directly injecting antagomir to wound edge was used to reduce the expression of miR-155. We found wounds treated with miR-155 antagomir had an obvious defect in immune cells requirements, pro-inflammatory factors IL-1ß and TNF-α reduced while anti-inflammatory factor IL-10 increased. With treatment of miR-155 antagomir, the expression of α-smooth muscle actin (α-SMA), Col1 and Col3 at wound sites all reduced both from mRNA levels and protein expressions. Wounds injected with antagomir resulted in the structure improvement of collagen, the collagen fibers were more regularly arranged. Meanwhile the rate of healing did not change significantly. These results provide direct evidences that miR-155 play an important role in the pathogenesis of fibrosis and show that miR-155 antagomir has the potential therapy in prevention and reduction of skin fibrosis.
Asunto(s)
MicroARNs/genética , MicroARNs/metabolismo , Cicatrización de Heridas/genética , Cicatrización de Heridas/fisiología , Actinas/genética , Animales , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo III/genética , Regulación hacia Abajo , Fibrosis , Inflamación/prevención & control , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/antagonistas & inhibidores , ARN Mensajero/genética , ARN Mensajero/metabolismo , Piel/lesiones , Piel/metabolismo , Piel/patología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: To observe the effects of Angelica dahurica extracts on the biological characteristics of human dermal fibroblasts in vitro and to preliminary explore its possible therapeutic mechanism for wound healing. METHODS: The optimal concentration of Angelica dahurica extracts was identified by analysing of proliferation activity of human normal fibroblasts (Fb) that treated with different concentration of Angelica dahurica extracts through thiazole blue (MTT) colorimetric assay. Cell cycle, collagen I and collagen III mRNA levels of the optimal Angelica dahurica extracts treated Fb were detected by flow cytometry (FCM) and real-time PCR techniques. RESULTS: At concentrations of 5 × 10(-4) to 5 × 10(-2) g/L, the Angelica dahurica extracts significantly enhanced the proliferation of Fb. The most significant concentration was 5 × 10(-3) g/L (t = 5.79, P < 0.01), at which an increased percentage of G1 to S and S to G2 phase cells (t = 11.2, 5.69, 2.44, P < 0.05) as well as an increased level of collagen I (1.61 ± 0.26 vs. 1.00 ± 0.16) and collagen III mRNA (3.36 ± 0.40 vs. 1.00 ± 0.14) were obtained compared to the control group (t = 6.69, 7.64, P < 0.01). CONCLUSIONS: Angelica dahurica extracts can notably promote the proliferation of Fb and accelerating the cell cycle of Fb as well as up-regulating the expression of collagen I and collagen III, which may enhance the process of wound healing.
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Angelica/química , Dermis/citología , Fibroblastos/efectos de los fármacos , Extractos Vegetales/farmacología , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colágeno/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , HumanosRESUMEN
BACKGROUND AND PURPOSE: Sirtuin1 (SIRT1), the founding member of mammalian class III histone deacetylases, is reported to be a drug target involved in fibrotic diseases. However, whether it is an effective drug target in hypertrophic scar treatment is still not known. EXPERIMENTAL APPROACH: In the present study, we observed that SIRT1 localized to both the epidermis and the dermis of skin tissues by immunohistochemistry. After knock-down of SIRT1 by shRNA or up-regulating SIRT1 by resveratrol, the expression of α-SMA, Col1 and Col3 in fibroblasts were detected by western blots. A mouse excision wound healing model was used to observe the changes in collagen fibre associated with the different expression levels of SIRT1. KEY RESULTS: SIRT1 expression was inhibited in hypertrophic scar tissue. The down-regulation of SIRT1 resulted in an increased expression of α-SMA, Col1 and Col3 in hypertrophic scar-derived fibroblasts. In contrast, the up-regulation of SIRT1 not only inhibited the expression of α-SMA, Col1 and Col3 in hypertrophic scar-derived fibroblasts but also blocked the activation of TGFß1-induced normal skin-derived fibroblasts. In the mouse model of wound healing, the deletion of SIRT1 resulted in denser collagen fibres and a more disordered structure, whereas resveratrol treatment led to a more organized and thinner collagen fibre, which was similar to that observed during normal wound healing. CONCLUSIONS AND IMPLICATIONS: The results revealed that SIRT1 negatively regulates TGFß1-induced fibroblast activation and inhibits excessive scar formation and is, therefore, a promising drug target for hypertrophic scar formation.
Asunto(s)
Cicatriz Hipertrófica/tratamiento farmacológico , Cicatriz Hipertrófica/metabolismo , Sirtuina 1/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos BALB C , ARN Interferente Pequeño/genética , Resveratrol , Sirtuina 1/biosíntesis , Sirtuina 1/deficiencia , Sirtuina 1/genética , Estilbenos/farmacologíaRESUMEN
OBJECTIVE: To investigate the therapeutic effect of nanometer silver impregnated dressing on gunshot wounds after being immersed in brine and tapwater in rabbits. METHODS: Rabbits were randomly divided into two groups after receiving gunshot wounds in both lower limbs. In group 1, the wounded limbs on the experimental side were immersed in brine for 5 h; in group 2, the wounded limbs on experimental side were immersed in tapwater for 5 h. All the wounds were treated with nanometer silver impregnated dressing on the experimental sides, while those of the control sides were treated with vaseline dressing. Biopsy was done after 30 min and 1, 2, 3, 4, 5 h, respectively. RESULTS: In group 1, the onset of inflammation around the wounds of the experimental sides was delayed, the inflammatory response was less serious, and the wounds were dry with less exudation compared to the controls. The mean healing time of the entry wounds on experimental and control sides was (29.4 +/- 6.6) d and (36.3 +/- 6.0) d (P < 0.01), respectively, and that of the exit wounds on experimental and control sides was (20.1 +/- 6.0) d and (27.3 +/- 5.7) d (P < 0.01), respectively. In group 2, only one of the experimental wounds showed mild inflammation, while all of the control wounds showed serious inflammation with much exudation. The mean healing time of the entry wounds on experimentsides was (13.0 +/- 1.52) d, while that on control sides was (16.0 +/- 3.10) d (P < 0.01). The mean healing time of exit wounds on experimental sides was (11.0 +/- 2.75) d, and those of the control sides was (15.6 +/- 2.85) d (P < 0.01). CONCLUSION: The nanometer silver impregnated dressing can control infection and accelerate healing in gunshot wounds in rabbits.
Asunto(s)
Infecciones Bacterianas/prevención & control , Vendajes , Plata/uso terapéutico , Heridas por Arma de Fuego/terapia , Animales , Infecciones Bacterianas/etiología , Femenino , Inmersión , Masculino , Modelos Animales , Nanotecnología , Conejos , Distribución Aleatoria , Sales (Química)/efectos adversos , Agua de Mar/efectos adversos , Plata/farmacología , Resultado del Tratamiento , Agua/efectos adversos , Cicatrización de Heridas/efectos de los fármacos , Heridas por Arma de Fuego/microbiología , Heridas por Arma de Fuego/patologíaRESUMEN
OBJECTIVE: To investigate the effect and the relevant potential mechanism of nonpeptide neurokinin 1 (NK1) receptor antagonist L-703,606 in the edema formation after burn injury. METHOD: L-703,606 treatment was performed in Sprague-Dawley (SD) rats at early stage after deep partial-thickness skin scalding. One hundred and fifty two adult male SD rats were used in the study and randomly divided into sham scald (SS, n=8), scald control (SC, n=48), and L-703,606 treatment (LT, n=48) groups. The rats in SC and LT groups were subjected to 20% total body surface area (TBSA) deep partial-thickness skin scalding. Modified Evans blue extravasation, tracing electron microscopy by lanthanum nitrate and mean water content assay were employed to observe and detect the changes of vascular permeability, ultrastructure and edema formation in adjacent tissue to the wounds and in the jejuna of rats at early stage (72 h) after scald. RESULTS: The pathological increase of vascular permeability in the periwound tissue and jejunum of rats in LT group were significantly lower than that in SC group (P<0.01), and recuperated earlier. Meanwhile, the changes of water contents of corresponding tissues in LT group were lighter than those in SC group (P<0.01). The ultrastructural changes of the microvessels in the peri-wound tissue of LT group showed that the junctions between microvascular endothelium cells were more narrow than those of SC group, moreover, and the number of opening and the engorgement and cavitation of the vascular endothelium cells decreased, the areosis and edema in perivascular tissue lightened, and the precipitation of the high eletron density lanthanum tracing agent in the interspace of the tissue decreased significantly in LT group. CONCLUSIONS: It is concluded that nonpeptide NK1-receptor antagonist L-703,606 could lighten the vascular permeability and edema formation in the periwound tissue and jejunum, and accelerate the normalization process of pathological changes in the tissues of rats after scald.
Asunto(s)
Quemaduras/patología , Edema/patología , Antagonistas del Receptor de Neuroquinina-1/farmacología , Quinuclidinas/farmacología , Receptores de Neuroquinina-1/metabolismo , Piel/lesiones , Animales , Agua Corporal/efectos de los fármacos , Permeabilidad Capilar/efectos de los fármacos , Yeyuno/efectos de los fármacos , Yeyuno/patología , Masculino , Microscopía Electrónica de Transmisión , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Piel/citología , Piel/patologíaRESUMEN
OBJECTIVE: To construct the tissue engineering seed cell (HaCaT cell line) with stable expression of the human epidermal growth factor (EGF), and analyze the changes of its biological characteristics. METHODS: PCDNA3.1-EGF eukaryotic expression vector was transferred into HaCaT cell, and G418 was utilized to select the HaCaT-EGF cell line. Using an inverted microscope, PCR, ELISA method to detect the changes of the cell morphology, the expression of the EGF gene and protein, and the mRNA expression levels of apoptosis related molecule Caspase-3, the cell cycle related protein cyclin D1. RESULTS: The mRNA expression levels of the obtained HaCaT-EGF cell were more than 100 times higher than the level of ordinary HaCaT cell. The colony of the HaCaT-EGF cells was more focused and tight compared to the empty vector transfected HaCaT cells and normal HaCaT cells. The expression levels of apoptotic factor Caspase-3 and cyclin D1 in HaCaT-EGF cell were significantly higher than those in the empty vector HaCaT- pcDNA3.1 cell, and the differences were statistically significant (P<0.01), but there was no significant difference compared to the normal HaCaT cells (P>0.05). CONCLUSIONS: HaCaT-EGF cell can continuously secrete EGF, and the biological characteristic is stable. It can be used for tissue engineering experiment and is an ideal seed cell for constructing tissue engineered skin.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Línea Celular/patología , Factor de Crecimiento Epidérmico/metabolismo , Queratinocitos/patología , Piel Artificial , Ingeniería de Tejidos , Cicatrización de Heridas , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica , Humanos , Queratinocitos/citología , Reacción en Cadena de la Polimerasa , ARN Mensajero , Fenómenos Fisiológicos de la Piel , Trasplante de Piel , Ingeniería de Tejidos/métodos , TransfecciónRESUMEN
Scarring, tightly associated with fibrosis, is a significant symptomatic clinical problem. Interleukin 10 (IL-10) has been identified as a candidate scar-improving therapy based on preclinical studies. However, the molecular mechanism of IL-10 in scar improvement is still uncertain. In this study, human dermal fibroblasts stimulated with TGF-ß1 were treated with IL-10 to analyze the mRNA and some of proteins' expression levels of type I collagen (Col1), type III collagen (Col3), alpha-smooth muscle actin (α-SMA), matrix metalloproteinase-1 (MMP1), MMP2, MMP8 and tissue inhibitor of metalloproteinase 1 (TIMP1), TIMP2 by real-time PCR and Western blot, to observe α-SMA-positive fibroblasts by immunocytochemistry. The contracture and improvement of fibroblast-populated collagen lattice (FPCL) and a murine model of wound healing were used to evaluate the scar-improving effects by histological staining. The results showed that IL-10 can significantly down-regulate the mRNA and protein expression levels of Col1, Col3, α-SMA, and up-regulate the mRNA expression levels of MMP1 and MMP8, and decrease α-SMA-positive fibroblasts. FPCL analysis showed that the IL-10 (20 ng/ml) can significantly inhibit the contracture, improve the architecture of FPCL. Wounds injected with IL-10 demonstrated that the appearance of scar was improved, the wound margin of scarring was narrow, and the deposition of collagens (Col1 and Col3) in regenerated tissue was relieved. These results provide direct evidences that IL-10 has the inhibitory effects on the excessive deposition of extracellular matrix components and fibroblast-to-myofibroblast transition, and show that IL-10 has the potential therapy in prevention and reduction of skin scarring.
Asunto(s)
Cicatriz/prevención & control , Dermis/patología , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Interleucina-10/farmacología , Interleucina-10/uso terapéutico , Factor de Crecimiento Transformador beta1/efectos adversos , Actinas/metabolismo , Animales , Células Cultivadas , Cicatriz/inducido químicamente , Cicatriz/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Dermis/efectos de los fármacos , Dermis/metabolismo , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Fibrosis , Humanos , Técnicas In Vitro , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 8 de la Matriz/metabolismo , Ratones , Ratones Endogámicos BALB C , Factor de Crecimiento Transformador beta1/farmacología , Cicatrización de Heridas/efectos de los fármacosRESUMEN
OBJECTIVE: To observe the effects of myrrh extract on biological characteristics of human dermal fibroblasts (Fb), and to explore its possible mechanisms in promoting wound healing. METHODS: Normal Fb was isolated from human foreskin tissue and cultured in vitro. The third to fifth passages of Fb were used in the experiment. (1) Fb were planted onto 96-well plate and divided into control group, and 1 × 10(-4), 1 × 10(-3), 1 × 10(-2), 1 × 10(-1), 1, 10, 1 × 10(2) g/L myrrh water extract groups and myrrh ethanol extract groups according to the random number table. Fb in control group were cultured with DMEM medium containing 0.25% calf serum (briefly called low-concentration serum medium), and those in various concentrations of myrrh water extract and myrrh ethanol extract groups respectively with low-concentration serum medium containing corresponding concentration of 2 kinds of myrrh extract. After being cultured for 48 h, cell morphology was observed with inverted-phase contrast microscope, and Fb proliferation activity (denoted as absorbance value) was determined with MTT method. (2) Fb were respectively planted into flasks and dishes and divided into two groups according to the random number table. Fb in control group were cultured with low-concentration serum medium, and that in 1 g/L myrrh water extract group with low-concentration serum medium containing 1 g/L myrrh water extract. After being cultured for 72 h, Fb cell cycle and the type I and III collagen mRNA expression were respectively determined by flow cytometry and real-time fluorescent quantitative PCR. Data were processed with LSD-t test. RESULTS: (1) Fb in all groups grew in long-spindle shape, but the cell fusion was much obvious in 1 g/L myrrh water extract group than in control group. Fb absorbance value in 1 × 10(-3), 1 × 10(-2), 1 × 10(-1), 1, 10 g/L myrrh water extract groups was respectively 0.378 ± 0.032, 0.402 ± 0.007, 0.390 ± 0.038, 0.453 ± 0.036, 0.390 ± 0.037, all higher than that in control group (0.332 ± 0.044, with t value respectively 2.24, 2.93, 2.69, 5.73, 2.71, P values all below 0.05). Compared with that in control group, Fb absorbance value in 1 × 10(-4) g/L myrrh water extract group was not statistically different (0.312 ± 0.048, t = 2.84, P > 0.05), while that in 1 × 10(2) g/L myrrh water extract group was significantly lower (0.154 ± 0.009, t = 7.17, P < 0.05). Fb absorbance values in 1 × 10(-3), 1 × 10(-1), 1, 10, 1 × 10(2) g/L myrrh ethanol extract groups were significantly lower than that in control group (with t values from 2.30 to 24.79, P values all below 0.05). (2) Compared with those in control group [(82.2 ± 7.9)% and (13.3 ± 2.3)%, (4.5 ± 0.8)%], the percentage of cells in G0/G1 phase in 1 g/L myrrh water extract group was obviously decreased [(74.3 ± 6.3)%, t = 6.77, P < 0.05], while those in S and G2/M phases increased [(16.6 ± 3.4)%, (9.1 ± 1.6)%, with t value respectively 7.53, 6.34, P values below 0.05]. Compared with those in control group (1.00 ± 0.05, 1.00 ± 0.06), the mRNA level of collagen III in 1 g/L myrrh water extract group was significantly up-regulated (1.38 ± 0.12, t = 3.81, P < 0.01), while that of collagen I was not statistically different (0.89 ± 0.08, t = 1.17, P > 0.05). CONCLUSIONS: Myrrh water extract can notably promote the proliferation of Fb, accelerate the cell cycle of Fb, and up-regulate the mRNA expression of type III collagen in Fb, which may be related to its mechanisms in promoting wound healing.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular , Colágeno Tipo III/metabolismo , Fibroblastos/citología , Humanos , Extractos Vegetales/farmacología , ARN Mensajero/genética , Terpenos/farmacologíaRESUMEN
Autophagy is a tightly regulated physiological process essential for cellular maintenance, differentiation, development, and homeostasis. Aberration of this process associates with the pathogeneses of several diseases in mammals. Hypertrophic scar (HS) is characterized by an abundance of collagenous tissue with hypercellularity. However, the molecular mechanism in HS formation is poorly understood. We compared the autophagic capacity in HS and its normal skin (NS) counterparts and explored the molecular mechanism of autophagy during the formation of HS. Microtubule-associated protein 1 light chain 3 (LC3) proteins in HS and NS were detected by immunohistochemistry, Western blot and quantitative real-time PCR (qPCR). The data showed that LC3 positive staining in HS was less intensive relative to NS group (p < 0.05). Three forms of LC3, with molecular weights of about 19 kDa (proLC3), 18 kDa (LC3-I) and 16 kDa (LC3-II), respectively, expressed in NS by Western blot. In contrast, only proLC3 expressed while both LC3-I and LC3-II were significantly downregulated in HS. The protein level of beclin 1 in HS was significantly lower compared with NS (p < 0.05). LC3 and beclin 1 mRNA levels in HS were significantly lower than that in NS (p < 0.05). These results suggest that the generation of LC3-I and LC3-II are interrupted in HS, and that the resultant decrease of autophagic capacity may associate with the pathogenesis of HS.
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Cicatriz Hipertrófica/metabolismo , Proteínas Asociadas a Microtúbulos/biosíntesis , Adolescente , Adulto , Proteínas Reguladoras de la Apoptosis/análisis , Autofagia/fisiología , Beclina-1 , Niño , Humanos , Proteínas de la Membrana/análisis , Persona de Mediana Edad , Adulto JovenRESUMEN
This study aimed to investigate the feasibility of using an immortal keratinocyte cell line, HaCaT cells, to effectively deliver epidermal growth factor (EGF) in a skin substitute to treat burn wounds. The skin equivalent was constructed with human EGF (hEGF) gene modified HaCaT cells obtained through stable gene transfection; these were applied to full thickness burn wounds in a rat model. The results showed that the hEGF gene modified HaCaT cells produced more than 390ng/l of bioactive hEGF in the culture supernatant. K19 and integrin-ß1 as keratinocyte differentiation markers were elevated in the hEGF gene modified HaCaT cells which were shown to be non-tumorigenic. The skin equivalent constructed with hEGF gene modified HaCaT cells demonstrated improved epidermal morphogenesis with a thick and compact epidermis. Wound healing was accelerated noticeably when applied with this skin substitute seeded with hEGF gene modified HaCaT cells in vivo. The results suggest that HaCaT cells modified with hEGF gene might be promising seed cells for construction of genetically modified skin substitute which can effectively secrete hEGF to accelerate wound repair and regeneration.
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Quemaduras/cirugía , Factor de Crecimiento Epidérmico/genética , Epidermis/fisiología , Técnicas de Transferencia de Gen , Queratinocitos/trasplante , Ingeniería de Tejidos/métodos , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Factor de Crecimiento Epidérmico/metabolismo , Estudios de Factibilidad , Humanos , Masculino , Ratones , Ratones Desnudos , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Thermal injury inhibits Akt activation and upregulates p38 mitogen-activated protein kinase, which in turn induces inflammation and increases apoptosis. This study aimed to elucidate the mechanism underlying the cytoprotective role of insulin in severe burns by examining the effects of insulin on inflammation and apoptosis mediated by p38 mitogen-activated protein kinase in burn serum-challenged cardiomyocytes. Neonatal rat cardiomyocytes were exposed to burn serum for 6 hours in the presence or absence of insulin and pretreated with inhibitors to p38 mitogen-activated protein kinase (SB203580) and Akt (LY294002). The authors examined expression of myocardial tumor necrosis factor-alpha, cardiac myofilament proteins caspase-3 and Bcl2, and apoptosis. Burn serum-induced upregulation of tumor necrosis factor was inhibited by both SB203580 and insulin. LY294002 reversed insulin-mediated downregulation of tumor necrosis factor. Both SB203580 and insulin inhibited apoptosis, resulting in fewer pyknotic nuclei and inhibition of caspase-3 activation and Bcl2 downregulation. LY294002 reversed insulin-mediated inhibition of apoptosis. Insulin decreases inflammatory cytokine expression and apoptosis via PI3K/Akt-mediated inhibition of p38 mitogen-activated protein kinase. The cytoprotective role of insulin suggests that it may have a potential role in strategies for treating thermal injuries.
Asunto(s)
Quemaduras/metabolismo , Insulina/farmacología , Miocitos Cardíacos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Análisis de Varianza , Animales , Apoptosis , Quemaduras/complicaciones , Quemaduras/patología , Caspasa 3/biosíntesis , Inhibidores de Caspasas , Modelos Animales de Enfermedad , Expresión Génica , Inflamación/prevención & control , Ratas , Transducción de Señal , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/biosíntesis , Proteínas Quinasas p38 Activadas por Mitógenos/biosíntesisRESUMEN
OBJECTIVE: To compare the difference of protein expression in the supernatant of heat injured keratinocytes (KC) and normal KC. METHODS: A model of heat injured KC was produced in vitro. The supernatant of normal KC and heat injured KC was collected after culture for 12 hours, and was ultrafiltered and lyophilized to get the protein. The protein sample was separated by immobilized pH gradient based two dimensional gel electrophoresis (2-DE). The gel was stained and the different expression of protein was analyzed using ImageMaster 2D analysis software. RESULTS: (1) Average protein spots were 1,898 +/- 113, 1,877 +/- 97 in the supernatant of normal and heat injured KC and 1,118 protein spots could be used for statistical analysis. (2) Statistical result showed that 26 protein spots were significantly different between the two groups. 16 protein spots were higher in the supernatant of normal KC and then 10 protein spots were lower in the normal group. (3) 16 protein spots, which included 10 kinds of proteins, were identified successfully as different spots. Lower expression proteins were alpha-enolase, actin cytoplasmic 2, peroxiredoxin-4, phosphoglycerate mutase 1, G protein-regulated inducer of neurite outgrowth l in the supernatant of heat injured KC. Higher expression proteins in heat KC were purine nucleoside phosphorylase, tumor necrosis factor ligand superfamily member 10, proteasome subunit alpha type-7, UDP-glucose 6-dehydrogenase in the supernatant of heat injured KC. CONCLUSIONS: The result indicated that there are some significant different expression proteins in the supernatant of normal KC and heat injured KC. These findings provide new data for screening major molecules of tissue repair and finding the mechanism of wound repair.
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Calor , Queratinocitos/metabolismo , Proteoma/metabolismo , Células Cultivadas , Electroforesis en Gel Bidimensional , Respuesta al Choque Térmico , HumanosRESUMEN
Keloids and hypertrophic scars are significant symptomatic clinical problems characterized by the excessive and abnormal deposition of collagen-based extracellular matrix (ECM) components. However, the molecular basis of keloid and hypertrophic scar formation has not been fully elucidated. Here, we demonstrated that down-regulation of the transcription factor Smad interacting protein 1 (SIP1) could be relevant to keloid and hypertrophic scar formation. The results of the present study show that the level of SIP1 mRNA is significantly decreased in pathological scar tissues and in normal skin and pathological scar fibroblasts treated with transforming growth factor ß1 (TGF-ß1). In contrast, the expression of SIP1 mRNA is not decreased in normotrophic scar samples. The SIP1 mRNA level inversely correlates with the mRNA level of type I collagen (COL1A2) and directly correlates with the mRNA level of matrix metalloproteinase-1 (MMP1). Overexpression of SIP1 in keloid and hypertrophic scar fibroblasts represses TGF-ß1-stimulated COL1A2 expression and induces MMP1 expression. Alternatively, knockdown of SIP1 in normal skin fibroblasts enhance TGF-ß1-induced COL1A2 levels. These findings suggest that SIP1 could be a regulator of skin fibrosis, and depletion of SIP1 in pathological scar tissues could result in an up-regulation of collagen and down-regulation of matrix metalloproteinase, leading to an abnormal accumulation of ECM along with fibrosis and pathological scar formation.
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Cicatriz/metabolismo , Fibrosis/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Queloide/metabolismo , Adolescente , Adulto , Anciano , Niño , Colágeno/metabolismo , Colágeno Tipo I , Regulación hacia Abajo , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Masculino , Metaloproteinasa 1 de la Matriz/metabolismo , Persona de Mediana Edad , ARN Mensajero/metabolismo , Proteínas de Unión al ARN , Piel/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Adulto JovenRESUMEN
AIM: To observe the difference in the expression of glucagon in normal and in diet-induced hyperlipidemic gestational rats. METHODS: Thirty-two female SD rats were divided into control group, hyperlipidemia group, gestation group and hyperlipidemic-gestation group. Fourteen days after gestation, real-time quantitative PCR was performed to detect the expression of preglucagon mRNA and Western blot was employed to detect the expression of glucagon in pancreas in different groups. RESULTS: The glucose peak of OGTT was 30 minutes after glucose load in all the rats. The insulin peak of OGTT was 15 minutes after glucose load in controls, while the insulin peak was 30 minutes after glucose load in other groups. Fasting insulin in both gestation rats and hyperlipidemic-gestation rats was higher than that in controls(21.68+/-2.55 vs 14.35+/-0.86 mIU/L, P<0.05; 25.76+/-3.31 vs 14.35+/-0.86 mIU/L, P<0.01). Insulin AUC in both hyperlipidemic-gestation rats and hyperlipidemia rats was also higher than that in controls(204.60+/-79.06 vs 129.71+/-11.33 mIU/L, P<0.05; 230.25+/-13.19 vs 129.71+/-11.33 mIU/L, P<0.05). PG relative mRNA levels in gestation rats and in hyperlipidemic-gestation rats were respectively 1.46 fold and 1.77 fold higher than that in controls (P<0.05 and P<0.01)and 1.54 fold higher than that in hyperlipidemia rats (P=0.01). Glucagon in gestation rats and hyperlipidemic-gestation rats was respectively 2.57 fold and 3.44 fold higher than that in controls (both P<0.01)and 2.9 fold higher than that in hyperlipidemia rats(P<0.01). CONCLUSION: Both hyperlipidemia and gestation may delay the releasing peak of insulin after glucose load. In normal gestation it is mainly reflected by fasting insulin resistance and in hyperlipidemic-gestation it is reflected by both fasting and glucose load insulin resistance. The rising pancreatic glucagons may partly lead to the increased gestational insulin resistance.
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Dieta/efectos adversos , Regulación de la Expresión Génica , Glucagón/genética , Hiperlipidemias/genética , Complicaciones del Embarazo/genética , Animales , Glucemia/metabolismo , Femenino , Prueba de Tolerancia a la Glucosa , Hiperlipidemias/sangre , Hiperlipidemias/inducido químicamente , Insulina/sangre , Resistencia a la Insulina , Páncreas/metabolismo , Reacción en Cadena de la Polimerasa , Embarazo , Complicaciones del Embarazo/sangre , ARN Mensajero/genética , ARN Mensajero/metabolismo , RatasRESUMEN
OBJECTIVE: To observe the effect of the supernatant of heat injured keratinocytes (KC) on biological behavior of the dermal fibroblasts (Fb). METHODS: Human dermal Fb were isolated and cultured. A model of heat injured KC (HaCaT) was reproduced in vitro. Supernatant of normal KC and the supernatant of KC culture 12 hours after heat injury were collected and diluted with non-serum DMEM in 1:1 volume ratio to make normal KC conditioned medium (NKCM) and heat injury KC conditioned medium (HKCM) respectively. Fb was respectively treated with non-serum DMEM and 2 kinds of conditioned medium. (1) The proliferation of Fb was detected with MTT method at post culture hour (PCH) 12, 24, 36, 48. (2) The apoptosis of Fb was determined by flow cytometry at PCH 12 (Fb were heat injured in advance; Fb without heat treatment was used as control). (3) At PCH 24, expression of a-SMA in Fb cytoplasm was determined with immunofluorescence method; expression of a-SMA mRNA in Fb was determined with real-time quantified PCR. Data were processed with one-way analysis of variance, and pairwise comparison among groups with LSD-t test. RESULTS: (1) The proliferation of Fb: the absorbance value of Fb cultured with HKCM at PCH 12, 24, 36, 48 was respectively higher than that of Fb cultured with non-serum DMEM (with t value respectively 1.89, 2.35, 2.02, 1.94, and P values all below 0.01). There were significant statistical differences between the absorbance values of Fb cultured with HKCM and those of Fb cultured with NKCM at PCH 12, 24, and 48 (at PCH 12, t = 1.83, P < 0.01; at PCH 24, t = 2.91, P < 0.05; at PCH 48, t = 1.83, P < 0.05). (2) Apoptosis of Fb cultured with HKCM was diminished as compared with that of Fb cultured with NKCM and of Fb without treatment (t = 3.31, P < 0.05; t = 1.47, P < 0.01). (3) The expression of alpha-SMA (red fluorescence) in Fb cultured with non-serum DMEM or NKCM was less as seen under fluorescence scope, and it was obviously increased in Fb cultured with HKCM. (4) The relative expression amount of alpha-SMA mRNA in Fb cultured with HKCM was 1.32 +/- 0.06, which was higher than that both in Fb cultured with NKCM (1.14 +/- 0.07, t = 2.51, P < 0.05) and in Fb cultured with non-serum DMEM (1.00 +/- 0.09, t = 1.77, P < 0.05). CONCLUSIONS: The supernatant of KC 12 hours after heat injury can obviously promote the proliferation of Fb, inhibit its apoptosis and accelerate transdifferentiation of Fb to myofibroblasts.
Asunto(s)
Medios de Cultivo Condicionados/farmacología , Fibroblastos/citología , Fibroblastos/metabolismo , Queratinocitos/citología , Actinas/metabolismo , Apoptosis , Diferenciación Celular , Células Cultivadas , Fibroblastos/efectos de los fármacos , Citometría de Flujo , Trastornos de Estrés por Calor , Calor/efectos adversos , Humanos , ARN Mensajero/genéticaRESUMEN
OBJECTIVE: To reproduce a model of heat injured KC in vitro and explore its apoptosis rate of KC due to heat injury at different temperature. METHODS: Human KCs were cultured in vitro, and they were incubated at 37, 41, 43, 45, 48, and 51 degrees C respectively for 10 mins in water bath. Trypan blue staining and Hoechst 33258 fluorescence staining were used respectively to determine necrosis and apoptosis of KC. Rates of apoptosis and necrosis of KC were analyzed quantitatively by flow cytometer. The proliferation activity of KC after heat injury was detected by MTT test. RESULTS: The results of trypan blue staining, Hoechst 33258 fluorescence staining, and flow cytometer demonstrated that number of apoptotic and necrotic KC increased gradually along with a rise of water bath temperature. The rates of apoptosis and necrosis of KC were respectively (12.3 +/- 3.2)% and (14.1 +/- 1.6)% at 45 degrees C, (27.7 +/- 5.1)% and (58.0 +/- 4.2)% at 48 degrees C. Rate of KC necrosis reached up to (83.0 +/- 5.3)% at 51 degrees C. Inhibition of KC growth reached a stationary phase when the injurious temperature reached 45 degrees C as observed with MTT test. CONCLUSIONS: Heat injury can induce apoptosis and growth inhibition of KC in vitro. Incubating KC at 45 degrees C for 10 mins is a good condition to reproduce a model of heat injured KC in vitro. This model may be used to study the biological character and apoptosis of KC after burn injury.
Asunto(s)
Apoptosis , Supervivencia Celular , Calor , Queratinocitos/citología , Quemaduras , Proliferación Celular , Células Cultivadas , Citometría de Flujo , HumanosRESUMEN
OBJECTIVE: To investigate the protective effect of melatonin on residual hair follicle cells of scald rats at early stage. METHODS: Eighteen male Sprague-Dawley rats were randomly divided into scald group, treatment group, sham group , with 6 rats in each group. The rats in scald group and treatment group were subjected to 30% TBSA partial thickness scald on the back, and were resuscitated with balanced solution after 1 hour, while those in sham group were immersed in water at 37 degrees C for 25 s to simulate scald, and did not receive fluid replacement. Rats in treatment group were intraperitoneally injected with 10 mg/kg melatonin solution at 1 minute, 8 hours and 12 hours after scald, while those in sham group and scald group were given equal volume of 1% alcohol sodium-isotonic saline instead. Tissue samples were harvested at 6, 12 and 24 post scald hours (PSH) for determination of MDA and GSH levels. Apoptosis of residul hair follicle was detected by TUNEL method and immunohistochemistry of caspase-3. RESULTS: The level of MDA in scald group at each time point was much higher than that in sham group (P < 0.01) and treatment group (P < 0.05), and it peaked at 12 PSH. The changes in GSH were just opposite to that of MDA. Under fluorescence microscope, the residual hair follicle cells were blue, and the apoptotic cells appeared green. The apoptosis rate in scald group at 6, 12, 24 PSH was obviously higher than that in sham (P < 0.01) and treatment groups (P < 0.05), which was (20.2 +/- 3.4)% vs (4.3 +/- 2.3)% vs (10.9 +/- 3.2)%, (31.2 +/- 3.6)% vs (5.1 +/- 2.5)% vs (19.1 +/- 3.7)%, (22.4 +/- 2.7)% vs (4.1 +/- 2.4)% vs (13.1 +/- 3.4)%, respectively. The score of caspase-3 positive cell in scald group was higher than those in sham group (P < 0.01) and treatment group (P < 0.05). CONCLUSIONS: There is obvious correlation between oxidative stress and apoptosis rate of hair follicle cells in rats with partial thickness scald. Early administration of melatonin may have anti-apoptosis ability for residual hair follicle cells by attenuation of oxidative stress.
Asunto(s)
Quemaduras/tratamiento farmacológico , Quemaduras/metabolismo , Folículo Piloso/metabolismo , Melatonina/uso terapéutico , Estrés Oxidativo , Animales , Apoptosis , Folículo Piloso/citología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: To study the effect of insulin in different concentrations on secretion function of growth factors of adipose-derived stem cells (ADSCs). METHODS: ADSCs were isolated from human abdominal adipose tissue and cultured. The immunophenotype and adipose induced-differentiation were identified, and the third generation cells were collected. The collected cells were assigned to 1 x 10(-8), 1 x 10(-7), 1 x 10(-6) mol/L insulin groups according to the concentration of added insulin. When cells grew into 70% confluence in conventional medium, ADSCs were cultured further in serum-free DMEM containing insulin in different concentrations for 3 days. ADSCs cultured in medium without insulin were used as control group. Secretion amount of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) of ADSCs were determined by enzyme-linked immunosorbent assay. The effects of the supernatant fluid of ADSCs' nutrient solution on the proliferation and collagen synthesis of the cultured fibroblast were detected by MTT chromatometry and hydroxyproline chromatometry. RESULTS: The secretion amounts of VEGF and HGF of ADSCs in 1 x 10(-8) and 1 x 10(-7) mol/L insulin groups [(471 +/- 41, 762 +/- 66 ng/L), (643 +/- 64, 930 +/- 67 ng/L), respectively] were significantly higher as compared with those in control group (286 +/- 47, 577 +/- 84 ng/L) (P < 0.05 or P < 0.01). No change occurred in the secretion amount of VEGF and HGF of ADSCs in 1 x l0(-6) mol/L insulin group (P > 0.05). The supernatant fluid of ADSCs' nutrient medium of 1 x 10(-8), 1 x 10(-7) mol/L insulin groups showed obvious stimulative effect on the proliferation and collagen synthesis of fibroblasts, and it was most obvious in the 1 x 10(-7) mol/L group (P < 0.05 or P < 0.01). CONCLUSIONS: Insulin in the concentrations of 1 x 10(-8) and 1 x 10(-7) mol/L can notably promote ADSCs' function of secreting VEGF and HGF.
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Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Insulina/farmacología , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Adipocitos/citología , Células Cultivadas , Fibroblastos/citología , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Células Madre/citología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVE: To investigate the feasibility of fabricating tissue engineering skin with human hair follicle bulge cells (HFBCs) to repair full-thickness skin wound. METHODS: HFBCs and dermal papilla cells (DPCs) isolated from human fetal hair follicles by collagenase digestion were cultured, purified and passaged. PGA-collagen scaffolds as bioengineered dermis were randomly divided into A and B groups. The HFBCs and DPCs (1 : 2) were seeded in scaffolds of group A and the equal amount of DPCs was seeded in scaffolds of group B as control. Then the keratinocyte sheets were seeded onto the surfaces of the scaffolds as bioengineered epidermis. The tissue engineering skins were then transplanted to repair the full-thickness wound on the back of nude mice. The wound healing process was observed and the plant histological changes of the transplanted engineered skin was observed with light microscope on 2, 4, 6 post-operation weeks (POW). RESULTS: The full-thickness defect of nude mice in A and B groups could be effectively repaired by bioengineered skins. On 2 POW, integral epidermal and dermal structures were observed in the wounds in A and B groups, with thin epithelial layer and basement membrane. On 4 POW, epithelial layer became thickening and rete pegs formation was observed in basement membrane in A group, but only thickening of epithelial layer was observed in B group. On 6 POW, rete pegs structure was seen to descend and hair-follicle-like structure was formed, while only thickened epithelial layer with flat basement membrane were formed in B group. CONCLUSION: From the composite skin engineered with PGA-collagen hybrid scaffolds and keratinocytes, HFBCs and DPCs could effectively repair the full-thickness skin defect of nude mice. The hair follicle stem cells participate in the process of anatomic repair of wound, and might be able to induce the repair of skin structure and function.
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Folículo Piloso/citología , Piel Artificial , Ingeniería de Tejidos/métodos , Animales , Técnicas de Cultivo de Célula , Células Cultivadas , Dermis/citología , Feto/citología , Humanos , Masculino , Ratones , Ratones Desnudos , Trasplante de PielRESUMEN
OBJECTIVE: To investigate the protective effect of insulin on oxygen-radical induced hepatic injury in severely scalded rats in early stage of severe scald. METHODS: Eighty-four male Sprague-Dawley rats were randomly divided into three groups: i. e, normal group, saline group, and insulin group, with 28 rat in each group. The rats in the latter two groups were subjected to 30% TBSA full-thickness scald on the back, and received intra-peritoneal injection of 40ml/kg isotonic saline, and subcutaneous injection of 3 IU/kg insulin, respectively. The total anti-oxygen capability (T-AOC), the expression of superoxide dismutase (SOD), reactive oxygen species (ROS) and intercellular adhesion molecule (ICAM-1) in hepatic tissue, and serum alanine transaminase (ALT) were determined in each group at 6, 12, 24, 48 post-scald hours (PSH) with corresponding methods. RESULTS: The hepatic T-AOC and SOD content were obviously decreased, while the ROS content were markedly increased at 6 PSH in saline group compared with that in normal group (P < 0.05 or P < 0.01). The expression of ICAM-1 and serum content of ALT were significantly higher than that in normal group at 12 PSH and 48 PSH (P < 0.01). At 24 PSH, the hepatic T-AOC (386 +/- 75) U/g and SOD content (210 +/- 39 ) U/g were obviously higher in insulin group than those in saline group [(124 +/- 18), (111 +/- 9) U/g, respectively, P < 0.01), but the ROS content (154 +/- 29 ) U/g was much lower than that in saline group [(351 +/- 41) U/g, respectively, P < 0.01]. At 48 PSH, the serum content of ALT and hepatic expression of ICAM-1 in insulin group exhibited obvious difference when compared with those in saline group (P < 0.01). Meanwhile, Pathological examination showed that hepatic injury was alleviated by insulin administration after scald. CONCLUSION: Insulin administration early after severe scald exhibits protective effect on liver function by improving anti-oxygen radical ability of rat liver.