Exposure to ultraviolet (UV) irradiation has detrimental effects on skin accompanied by the increased metabolism of hyaluronan (HA), a linear polysaccharide important for the normal physiological functions of skin. In this study, the modulation of human keratinocyte response to UVB irradiation by HA (970 kDa) was investigated. Immortalized human keratinocytes (HaCaT) were irradiated by a single dose of UVB and immediately treated with HA for 6 and 24 h. The irradiation induced a significant decrease in the gene expression of CD44 and toll-like receptor 2 6 h after irradiation. The expressions of other HA receptors, including toll-like receptor 4 and the receptor for HA-mediated motility, were not detected in either the control or UVB-irradiated or HA-treated HaCaT cells. UVB irradiation induced a significant decrease in the gene expression of HA synthase-2 and hyaluronidase-2 6 h after irradiation. The expressions of HA synthase-3 and hyaluronidase-3 were not significantly modulated by UV irradiation. Interestingly, HA treatment did not significantly modulate any of these effects. In contrast, HA significantly suppressed UVB-induced pro-inflammatory cytokine release including interleukin-6 and interleukin-8. Similarly, HA treatment reduced the UVB-mediated production of transforming growth factor β1. HA treatment also significantly reduced the UV irradiation-mediated release of soluble CD44 into the media. Finally, HA partially, but significantly, suppressed the UVB-induced decrease in cell viability. Data indicate that HA had significant protective effects for HaCaT cells against UVB irradiation.
- MeSH
- antigeny CD44 biosyntéza genetika MeSH
- exprese genu MeSH
- glukuronosyltransferasa biosyntéza genetika MeSH
- hyaluronoglukosaminidasa biosyntéza genetika MeSH
- interleukin-6 biosyntéza MeSH
- interleukin-8 biosyntéza MeSH
- keratinocyty účinky léků metabolismus účinky záření MeSH
- kůže účinky léků metabolismus účinky záření MeSH
- kyselina hyaluronová metabolismus farmakologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- toll-like receptor 2 biosyntéza genetika MeSH
- transformující růstový faktor beta biosyntéza MeSH
- ultrafialové záření MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVES: Determination of cell numbers is a crucial step in studies focused on cytokinetics and cell toxicity. The impedance-based analysis employing electronic sensor array system xCELLigence System allowing label-free dynamic monitoring of relative viable adherent cell amounts was compared with the most utilized methods for relative quantification of viable cell numbers based on a determination of cellular metabolism. DESIGN: Colorimetric assay based on reduction of tetrazolium salt (MTT) by mitochondrial enzymes and chemiluminiscent assay based on intracellular adenosine triphosphate (ATP) determination were compared with the impedance-based system. Cell morphology was compared by microscopic evaluation. Normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF), together with 3T3 mouse fibroblast and HaCaT keratinocyte cell lines were employed. RESULTS: The progress of cell growth curves obtained by different methods during 72 hours reflected cell type and cell seeding densities. The impedance-based method was found to be applicable for the determination of the cell proliferation of 3T3 fibroblasts, HaCaT and NHDF, since the comparison of this method with ATP and MTT determinations showed a comparable results. In contrast, the proliferation of NHEK measured by the impedance-based method did not correlate with other methodological approaches. This could be accounted to the specific morphological appearance of these cells. CONCLUSION: The study shows the impedance-based detection of viable adherent cells is a valuable approach for cytokinetics and pharmacological studies. However, the specific morphological characteristics of cell lines have to be considered employing this method for determination of cell proliferation without using other reference methods.
- MeSH
- adenosintrifosfát metabolismus MeSH
- buněčné linie MeSH
- buňky 3T3 MeSH
- časové faktory MeSH
- elektrická impedance MeSH
- fibroblasty cytologie MeSH
- keratinocyty cytologie MeSH
- kolorimetrie MeSH
- kultivované buňky MeSH
- lidé MeSH
- luminiscenční měření MeSH
- mitochondrie enzymologie metabolismus MeSH
- myši MeSH
- oxidace-redukce MeSH
- počet buněk metody MeSH
- proliferace buněk MeSH
- škára cytologie MeSH
- tetrazoliové soli metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
