Influences On The Cells Morphology, Population And Expression Of INOS In Normal Human Keratinocytes And Fibroblasts By UVA Irradiation

ObjectiveTo compare the effects of UVA irradiation on cell morphology, population, expression of inducible nitric oxide synthase (iNOS) of keratinocytes with that of human fibroblasts . The purpose of this study was to probe the photodamage in keratinocytes and fibroblasts induced by ultraviolet A radiation, and to investigate biological effectiveness of iNOS during the photodamage. That may supply experimental evidences for UVA protection and phototherapy.Methods1. Primary normal human keratinocytes and fibroblasts were cultured. Cells were divided into four groups: keratinocytes irradiation group, fibroblasts irradiation group, keratinocytes control group and fibroblasts control group. The two irradiation groups accepted 5 J/cm~2 UVA irradiation. Sequentially, cells were culture continuely for 24h, 48h and 72h after irradiation. Then cells morphology were directly observed under inverted microscope and with HE stained. Live cell number was accounted by Trypan blue staining.2. The expression of iNOS mRNA in keratinocytes and fibroblasts after irradiation by UVA was detected by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR).3. The expression of iNOS protein in keratinocytes and fibroblasts after irradiation by UVA was identified by western blot.Results1. Cell morphology and population: There was no change on morphology in keratinocyte at three time points after 5J/cm~2 UVA irradiation. Cell population began to decrease at 48h after irradiation than before (p<0.01). There was no significant difference between 48h and 72h(p>0.05 ). Fibroblast morphology had appeared shrinkage at 24h after 5 J/cm~2 UVA irradiation. Cell population decreased at 24h after irradiation than before (p<0.01) , but there was no significant difference among the three time points (p>0.05 ) . However, there was no change in the control on morphology. It was duplicative tendency on cell population. 2. iNOS mRNA and protein: iNOS mRNA and protein expression of keratinocyte and fibroblast weren't detected on control group. iNOS expession of keratinocyte reached the peak at 48h ,and significantly different compared to that at 24h (p<0.01), but we didn't detect them at 72h after exposure. However, in fibroblast, iNOS expession reached the peak at 24h, and significantly different compared to that at 48h and 72h. But there was no significant difference between 48h and 72h (p>0.05).Conclusion1. Photodamage of fibroblasts induced by UVA appeared more easily and lasted for longer time than keratinocytes.2. iNOS expression in keratinocytes and fibroblasts can be induced by UVA. iNOS expression in fibroblasts reached the peak earlier and lasted for longer time than keratinocytes.2. iNOS was related to photodamage induced by UVA.