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Primary Human Keratinocytes (primary + human_keratinocyte)
Selected AbstractsEstablishment and characterization of immortalized human gingival keratinocyte cell linesJOURNAL OF PERIODONTAL RESEARCH, Issue 6 2008S. Gröger Background and Objective:, Primary human keratinocytes are used to analyze the properties of the oral epithelium and the early stages of oral bacterial infections. In vitro, these cells are characterized by their short life span and restricted availability. Approaches for culturing these cells will end after approximately 6,10 passages as a result of entry into apoptosis. For this reason, it is important to generate cell lines suitable for obtaining an unlimited source of cells. Therefore, the aim of the present study was to generate gingival keratinocyte cell lines and to compare their in vitro behaviour with those of primary human gingival keratinocytes. Material and Methods:, Primary human gingival keratinocytes were immortalized with a combination of the human papilloma virus onkoproteins E6 and E7. The pattern of the cytokeratins, involucrin and filaggrin was investigated by intracellular staining using flow cytometry. This method allows quantitative analysis of the expression of a variety of intracellular or extracellular markers. Results:, The immortalized cell lines showed many morphological similarities, expressing a cytokeratin pattern that is comparable with that of primary gingival keratinocytes. Furthermore, they developed transepithelial electrical resistance, which is a marker for the generation of tight junctions. These results indicate that the cells might be able to act as an epithelial barrier, reflecting the reaction of primary human cells. Conclusion:, The establishment of a continuous line of human gingival epithelial cells with functional characteristics of the epithelial barrier provides a valuable in vitro model for using to study the early steps of gingival/periodontal infections. [source] Nucleofection: a new, highly efficient transfection method for primary human keratinocytes,EXPERIMENTAL DERMATOLOGY, Issue 4 2005Jörg H. W. Distler Abstract:, Transfection is an essential tool for numerous in vitro applications including studies of gene expression, promoter analysis, and intracellular signaling pathways and also for therapeutic strategies such as tissue engineering and gene therapy. However, transfection of primary cells including keratinocytes with common methods such as calcium phosphate, DEAE-dextran, liposome-mediated transfer, electroporation or viral vectors is problematic because of low transfection efficiency and the induction of terminal differentiation. Here we analyzed the use of nucleofection, a new, electroporation-based transfection method that enables the DNA to enter directly the nucleus, for the transfection of keratinocytes. Several different conditions were tested and optimized, resulting in a final transfection efficiency of 56% in primary human epidermal keratinocytes. This efficiency is superior to all non-viral transfection methods reported so far. The number of non-viable keratinocytes after nucleofection was low, varying between 14 and 16%. In contrast to other transfection protocols, nucleofection did not induce terminal differentiation in the transfected keratinocytes. In addition, nucleofection is a fast method, because the results can be analyzed within 7 h. In summary, nucleofection is a fast, easy and highly effective alternative for the transfection of primary human keratinocytes, which offers new opportunities for various research applications. [source] Antipsoriatic drug anthralin induces EGF receptor phosphorylation in keratinocytes: requirement for H2O2 generationEXPERIMENTAL DERMATOLOGY, Issue 2 2004Dominik Peus Abstract: Even though anthralin is a well-established topical therapeutic agent for psoriasis, little is known about its effects and biochemical mechanisms of signal transduction. In contrast to a previous report, we found that anthralin induced time- and concentration-dependent phosphorylation of epidermal growth factor receptor in primary human keratinocytes. Four lines of evidence show that this process is mediated by reactive oxygen species. First, we found that anthralin induces time-dependent generation of H2O2. Second, there is a correlation between a time-dependent increase in anthralin-induced epidermal growth factor receptor phosphorylation and H2O2 generation. Third, the structurally different antioxidants n -propyl gallate and N -acetylcysteine inhibited epidermal growth factor receptor phosphorylation induced by anthralin. Fourth, overexpression of catalase inhibited this process. The epidermal growth factor receptor-specific tyrosine kinase inhibitor PD153035 abrogated anthralin-induced epidermal growth factor receptor phosphorylation and activation of extracellular-regulated kinase 1/2. These findings establish the following sequence of events: (1) H2O2 generation, (2) epidermal growth factor receptor phosphorylation, and (3) extracellular-regulated kinase activation. Our data identify anthralin-induced reactive oxygen species and, more specifically, H2O2 as an important upstream mediator required for ligand-independent epidermal growth factor receptor phosphorylation and downstream signaling. [source] Nickel-induced keratinocyte proliferation and up-modulation of the keratinocyte growth factor receptor expressionEXPERIMENTAL DERMATOLOGY, Issue 4 2003Cinzia Marchese Abstract: Keratinocytes play a key role in the pathogenesis of allergic contact dermatitis (ADC) induced by the sensitizing agent nickel. We analyzed here the effects of treatment with nickel and of the pretreatment with zinc on HaCaT cells and primary human keratinocytes. Cell counting, 5-bromo-2,-deoxyuridine incorporation assay and adenosine triphosphate (ATP) bioluminescence detection showed that treatment with NiSO4 induced DNA synthesis and cell proliferation and that pretreatment with ZnSO4 was able to abrogate this proliferative effect. This nickel-induced cell growth appeared enhanced when primary human keratinocytes were co-cultured with fibroblasts. Western blot analysis demonstrated that nickel ions induced up-modulation of the expression of the keratinocyte growth factor receptors (KGFR) without affecting the keratinocyte differentiation, whereas the protein levels of the epidermal growth factor receptor (EGFR) and of its ligand transforming growth factor-alpha (TGF-,) appeared unmodified by the treatment. Double immunofluorescence showed that the effect of nickel on DNA synthesis was mainly exerted on KGFR expressing cells, suggesting that KGFR up-modulation could be required for the nickel-induced cell proliferation. These results indicate that KGFR and its ligands may play a role in the mechanism of action of nickel ions and in the protective effect of zinc pretreatment. [source] Ligand-independent Regulation of the hairless Promoter by Vitamin D Receptor,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2008Andrew Engelhard The characteristic alopecia associated with mutations in the hairless (hr) and vitamin D receptor (VDR) genes defines the resulting genetic disorders, known as atrichia and VDRRIIa rickets, as phenocopies. In both cases, the separation of the dermal papilla from the regressing hair follicle at the onset of the first catagen phase of the hair cycle and the development of dermal cysts and utricules subsequent to mutation of either gene suggests that their activities affect the same regulatory pathways. VDR functions as a hormonally activated transcription factor, and a role in transcription has been postulated for Hr due in part to its nuclear localization and homology with the GATA-1 zinc-finger domain. Therefore, we examined the hypothesis that VDR and Hr have a direct regulatory effect on each other via a transcriptional mechanism. Ectopic expression of the VDR repressed hr promoter activity in HaCaT cells and primary human keratinocytes (PHKs). While this repression occurs in the absence of 1,25 dihydroxyvitamin D3 (D3), the addition of ligand greatly augments the effect. However, we also demonstrate the rare phenomenon of ligand-independent promoter transactivation by VDR. We show that the full-length promoter is transactivated by VDR in a ligand-independent and cell type-specific manner, suggesting that direct transcriptional regulation of hr by the VDR accounts in part for the phenotypic overlap between atrichia and VDRRIIa rickets. [source] | ||||||||||