Effect On Cell Cycle Regulation After Ultraviolet Irradiation In Human Keratinocyte

Objectite To establish a mode of ultraviolet irradiated human keratinocytes(HaCaT cell line). Aim to investigate the effects of UVB on the procession of cell cycle and apoptosis. And observe the protective effects of VE against UVB radiation. And to study the expression of related gene p53, bcl-2 on hereinbefore condition. Methods By means of cell culture, MTT assay, flow cytomer and western blot analysis, we analyzed HaCaT cell survival ratio, various phases of cell cycle and apoptosis. At the same time, the effects of VE on this event were tested and the change in p53, bcl-2 protein expression level was observed. Results 1. The survival ratio of HaCaT keratinocytes decreased gradually after either being irradiated by UVB at different dosage and then incubated at the same duration or being irradiated at the same dosage and then incubated at various duration. The survival ratios of the cells irradiated at different dosage and then incubated for various duration would increase after arriving at the minimum point except the group irradiated for 15 min. 2. The proliferation indexes {(S+G2)/(G1+S+G2)} increased gradually with increasing culture time after cells were irradiated for less than 5 min.UVB resulted decrease in HaCaT cell viability of proliferation in dose- and time-dependent manner. 3. We found the apoptosis of normal HaCaT existing in not only morphologic but flow cytometry results. 4. The apoptosis ratio of HaCaT increased gradually with UVB dosage increasing and culture time prolonging. 5. Obvious protective effect of VE on the cells was observed against UVB irradiation-induced cell cycle arrest and apoptosis, with optimal dose of VE of 40 μg/ml. VE was pretreated or treated both at time before and after UVB radiation providing a better protection than posttreatment. 6. Flow cytometry analysis indicated that VE pretreatment suppressed both a decrease in PI and an increase about 50% in apoptotic cells by UVB exposure. 7. The expression of p53 and phospho-p53(Ser15) protein increased following UVB irradiation. The p53 protein level reached its peak at 12 h, and the expression of phosph-p53protein sustained 12h .But the gene expression of bcl-2 decreased, which remained low up until 12h following the UVB-irradiation. 8. VE treatment attenuated the UVB-induced the gene expression of p53 but elevated that of bcl-2. As the prolonging of the time the expression of bcl-2 protein decreased. No expression of phosph-p53 was detected. Conclusions UVB inhibit HaCaT human keratinocyte growth in a definite dose- and time- dependent manner. UVB exposure of HaCaT resulted in Gl cell cycle arrest and apoptosis, which is antagonized by VE. Changes of p53, phosph-p53 and bcl-2 expression might play role in DNA damage response induced by UVB irradiation. The expression of bcl-2 protein might contribute to the protective effect of UVB irradiation.