UV-induced Apoptosis In Human Lens Epithelial Cells And Its Mechanism In Vitro

ObjectiveTo explore the apoptosis-inducing effect of ultraviolet (UV) on human lens epithelial cell (HLEC) and its possible mechanism. MethodHLEC line was generously provided by Ophthalmology Research Institute of Oakland University USA. All the experimental groups were exposured to the same UV light source. The irradiance provided at 365nm (UVA) was 0.29mw/cm2 and at 315nm (UVB) was 0.09 mw/cm2. HLEC were divided into six groups according to UV radiation time:Omin group (control group),5min group,10min group,15min group and 30min group. Cells were returned to the CO2 incubator for a 12 hr period. Then cells were prepared for morphological,cytochemical and biochemical analyses at varying times after UV exposure.1. UV induced apoptosis in HLEC:Qualitative analysis on apoptosis of HLEC were performed by Hoechst 33342 staining, AO/EB staining and DNA ladder. Quantities analysis on apoptosis of HLEC were performed by AO/EB staining and flow cytometry analysis (FCA, Annexin?+PI staining); Also the relationgship between exposure dose and UV radiation-induced apoptosis was analyzed.2. UV-induced changes in gene expression and cell cycle:Bax and Bcl-2 expression changes in HLEC were detected by hybridization in situ. Changes in HLEC cell cycle was detected by FCA (PI staining).3. The influence of UV irradiation on ALDH1 of HLEC:Change of ALDH1 in HLEC was detected by immunohistochemistry staining and western blot. Result1. Hoechst 33342 staining and AO/EB staining revealed cells with characteristic morphological changes of apoptosis in all experimental groups, such as:cell shrinkage, nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation. DNA fragmentation assays showed there were typical DNA ladder in 10min,15min and 30min group except 5min group. All above confirmed apoptosis in HLEC after UV exposure. AO/EB staining showed apoptosis percentage were 1.82±0.53%?13.15±2.32%?17.58±1.62%?31.16±3.03%?29.25±2.53% in 0min,5min,1 Omin,15min and 30min group respectively (F=146.10, P<0.05). FCA showed apoptosis percentage were 1.98±0.84%?11.90±3.21%.16.15±3.05%.33.93±3.74%,22.72±6.05%in above groups respectively (F=34.16, P<0.05). Quantities analysis on apoptosis by both methods showed apoptosis percentage increased with UV exposure time.2. The expression level of Bax mRNA was increased with UV exposure time, reached peak at 15min and 30min. Whereas the expression level of Bcl-2 mRNA decrease with UV exposure time. The lowest was at 30min. There was significant differece between each group (P<0.05) Apoptosis percentage was negative correlation with Bcl-2/Bax ratio (r=-0.874, P<0.05).Cells of G2/M increased in experimental groups. The difference was significant when compared with control group (Omin group) (P<0.05). Cells of S phase decrease with UV exposure time (F=40.34, P0.05).3. Percentage of positive reaction cells of ALDH1 were 39.23±5.34%,30.57±4.45%,17.91±4.28%,10.25±3.01%?3.83±0.83% in 0min,5min,10min,15min and 30min group respectively. The intensity of immunohistochemistry staining and percentage of positive reaction cells decreased with UV exposure time (F=68.827, P<0.05). ALDH1 western blot also showed the same tendency of change (F=17.256, P0.05)?Conclusion1. UV can induce apoptosis in HLEC in vitro. Apoptosis percentage is positively co-related with UV dose and exposure time.2. UV irradiation can down-regulate bcl-2 and up-regulate Bax expression in HLEC.3. UV irradiation can induce cell cycle arrest of G2/M, it is also positively co-related with UV dose.4. ALDH1 in HLEC decreased with UV dose and exposure time.