الفهرس 
Material and MethodsOnly 14 pages are availabe for public view
 |  | from | 233 |  |  |
| | | from | 233 | | |
Abstract
This research was aimed at the identification, extraction, and characterisation of the chemokine (s) released from cultured human keratincytes following appropriate activiation. In one part of the study, UVB was employed as a stimulus, and in another part keratincytes were stimulated by incubating them with different cytokines individually used in combination. The chemokine proteins were identified using the Enzyme linked immunosorbent assay technique and then characterized by a combination of standard protein purification method and mass spectrometry.
The results of experiments detailed in the present study allow the following conclusions:
1- Low calcium serum free medium allowed clonal growth of keratincytes that are proliferating and non-differentiating and having basal morphology. Purifying keratincytes cultures from fibroblast and melaocyte contamination is essential to avoid misleading results.
2- Irradiating keratincytes with the dish lids on eliminated the effect of UVC and minimised the possibility of contamination of cell culture.
3- keratincytes play a central and eminent role in skin inflammation.
* The present study had shown that epidermal keratincytes of the skin exhibit the capacity to produce a variety of inflammatory mediators, both lipid and protein in nature, in purpose to the UVB light stimulus. These mediators are capable of mediating the vascular and cellular responses of these inflammatory reactions.
*The finding that keratincytes possess the capacity to express RANTES suggests that keratincytes might play a role in mediating cutaneous late phase reactions and dermatoses characterised by eosinophlic spongiosis.
Keratincytes through their capacity to secrete IL-8 and RANTES appear to play a role in inflammatory and immunological skin diseases.
4- The IL-8 protein released from UVB-irradiated human keratincytes corresponds to authentic IL-8 (72) Mr 8382.3.
5- Several factors affect the viability of keratincytes cultures, these include: UVB, Cytokines, indomethacin, and cyclohexamide. The major impact on keratincytes viability was observed when the irradiated cells incubated in media contained CHX 10Mg/ml. Normal human keratincytes appeared to be better protected against these factors than HaCaT cells.
6- it seems likely that UVB-irradiated keratincytes produce factors that mediate inflammation and at the same time produce factor (s) that protect them from or switch off this inflammation. Prostaglandins might be one of those factors and others, to be identified, require de novo protein synthesis.