RESUMEN
Rat liver myofibroblasts (MFB) were isolated by repeated passaging of nonparenchymal liver cell fraction. They were cultured on polystyrene Petri dishes, on fibrin or on type I collagen gels for 5 days. Quantitative RT-PCR, Western blotting, zymography and immunocytochemistry were used to study differences in cell morphology and protein expression. MFB were large and spread on plastic substrate, with prominent alpha-smooth muscle (alpha-SMA) fibres. They turned much smaller and elongated on collagen which was accompanied by the rearrangement of the cytoskeleton and a decrease in alpha-SMA and beta-actin content. Collagen gel induced the expression of a group of metalloproteinases (MMP-2, -3, -9, -13), on mRNA and protein level which resulted in the degradation of the gel. This response was accompanied by changes in the mRNA expression of cytokines of TGF-beta family, CTGF and interleukin-6, as well as of osteopontin and thrombospondin-2 that are involved in metalloproteinases (MMPs) regulation. The expression of MMPs substrates, collagen types I, IV and XII did not change or decreased. The effects of fibrin gels on MFB were milder than those of collagen. MFB assumed to deposit collagen and other ECM components in fibrotic liver, besides hepatic stellate cells, also possess a great collagenolytic potential.
Asunto(s)
Colágeno Tipo I/metabolismo , Colagenasas/metabolismo , Hígado/enzimología , Miofibroblastos/enzimología , Actinas/metabolismo , Animales , Biomarcadores/metabolismo , Western Blotting , Separación Celular/métodos , Forma de la Célula , Células Cultivadas , Colagenasas/genética , Citocinas/metabolismo , Citoesqueleto/enzimología , Fibrina/metabolismo , Inmunohistoquímica , Hígado/citología , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
AIM: To determine the response of dental pulp stem cells (DPSCs) to DNA-damaging cytostatic cisplatin and compare it with the response of normal human dermal fibroblasts (HDFs). METHODOLOGY: Dental pulp stem cells were exposed to 5, 10, 20 or 40 µmol L(-1) of cisplatin. The proliferation of affected cells was assessed by a Z2 Counter and viability was assessed by means of a Vi-Cell XR using Trypan blue exclusion staining. Cell cycle analysis and induction of apoptosis were performed by flow cytometry. Induction of apoptosis was determined by monitoring the activities of caspases. The expression of proteins was detected by electrophoresis and Western blotting. The descriptive statistics of the results was analyzed by Student's t-test. RESULTS: Dental pulp stem cells had a greater genotoxic stress response to cisplatin compared to HDFs. All three main Mitogen-activated protein kinases (MAPK) families - extracellular signal-regulated kinases (ERK), c-Jun-N-terminal kinase (JNK) and p38 were activated after treatment of DPSCs with cisplatin. The activation of MAPK pathways was not observed in HDFs exposed to cisplatin. The exposure of DPSCs and HDFs to cisplatin provoked an increase in p53 and p21 expression and p53 phosphorylation of serine 15. Higher concentrations of cisplatin reduced the viability of DPSCs and HDFs and induced the activation of caspases 3/7 and 9. CONCLUSION: Dental pulp stem cells had a greater genotoxic stress response to cisplatin compared to HDFs. Cisplatin in higher concentrations triggered activation of MAPK and apoptosis in DPSCs but not in HDFs.
Asunto(s)
Cisplatino/toxicidad , Citostáticos/toxicidad , Pulpa Dental/citología , Ectodermo/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Apoptosis/efectos de los fármacos , Caspasa 3/efectos de los fármacos , Caspasa 7/efectos de los fármacos , Caspasa 8/efectos de los fármacos , Caspasa 9/efectos de los fármacos , Técnicas de Cultivo de Célula , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/efectos de los fármacos , Pulpa Dental/efectos de los fármacos , Ectodermo/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mutágenos/toxicidad , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Serina/efectos de los fármacos , Piel/citología , Piel/efectos de los fármacos , Proteína p53 Supresora de Tumor/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/efectos de los fármacosRESUMEN
Cellular response to ionizing radiation-induced damage depends on the cell type and the ability to repair DNA damage. Some types of cells undergo apoptosis, whereas others induce a permanent cell cycle arrest and do not proliferate. Our study demonstrates two types of response of embryonic diploid fibroblasts WI-38 to ionizing radiation. In the WI-38 cells p53 is activated, protein p21 increases, but the cells are arrested in G2 phase of cell cycle. Some of the cells die by apoptosis, but in remaining viable cells p16 increases, senescence associated DNA-damage foci occur, and senescence-associated beta-galactosidase activity increases, which indicate stress-induced premature senescence.
Asunto(s)
Apoptosis/efectos de la radiación , Senescencia Celular/efectos de la radiación , Daño del ADN/efectos de la radiación , Diploidia , Células Madre Embrionarias/efectos de la radiación , Fibroblastos/efectos de la radiación , Apoptosis/genética , Línea Celular , Supervivencia Celular/genética , Supervivencia Celular/efectos de la radiación , Senescencia Celular/genética , Daño del ADN/genética , Células Madre Embrionarias/fisiología , Fibroblastos/fisiología , Humanos , Radiación IonizanteRESUMEN
BACKGROUND: Hyaluronan (HA) plays an important role in the repair of damaged skin and has been used for the treatment of wounds. Iodine is a mild topical antiseptic. AIM: A mixture of high molecular weight HA with the iodine complex KI(3) (hyiodine) was reported to accelerate wound healing in patients with diabetes and patients after surgery. We investigated how this mixture affects wound contraction, granulation tissue (GT) and wound edges in excision skin wounds in rats. METHODS: Hyiodine was applied to full-thickness wounds made on the back of rats. The areas of the contracting wounds were calculated from digital photographs. The moving edges of the wound were studied by histological methods. The properties of GT were studied in wounds in which contraction was prevented by the insertion of plastic rings. The effects of hyiodine were compared with those of high molecular weight (1200 kDa) HA, low molecular weight (11 kDa) HA and KI(3) solution. RESULTS: Hyiodine accelerated wound contraction significantly in the first 5 days of healing. On day 3, hyiodine-treated wounds had reduced to 63% of the original area, whereas the wound area in saline-treated animals was 75% of the original size. The proliferating epidermis was thicker in hyiodine-treated animals on day 7. In the wounds with inserted rings, hyiodine caused little change in GT, but the weight of the crust/exudate formed on the top of the wound was increased by 351% compared with only minor changes caused by the hyiodine components alone. CONCLUSIONS: Hyiodine supports wound healing by stimulating wound contraction and epidermal proliferation and by keeping the wound moist through increased exudation.