| ObjectiveIn recent years, ultraviolet light and human skin healthy relationship has been a hot research. UV is the "double-edged sword", on the one hand, ultraviolet light has been considered the killer of skin, in addition to a variety of photosensitive skin diseases may result, too much ultraviolet radiation is the most important cause of skin photoaging and exposed parts of skin cancer-prone factors; on the other hand, there are also UV beneficial biological effects, suitable ultraviolet radiation can improve immunity, prevent certain autoimmune diseases, promote bone growth and development, reduce the incidence of cancer. The existence of a consistent relationship between these beneficial biological effects may be related to low dose irradiation-induced hormesis. Adaptive response is an important part of the radiation hormesis. Our previous experiments have confirmed that low doses of ultraviolet A (UVA) radiation can induce adaptive response of cultured skin cells appear[1], this adaptive response manifested as single or multiple low-dose UVA irradiation can reduce the subsequent pre-lethal dose of UVA irradiation of cultured skin fibroblasts to toxicity on the morphology, and the proportion of apoptosis decreased to reduce and accelerate the repair of DNA strand breaks, but this adaptive response mechanism remains to be elucidated. Autophagy (autophagy) in eukaryotic cells is a common phenomenon of life, the cell is deformed, aging or damaged proteins and organelles transported to the lysosome digest degradation chamber a metabolic process. Autophagy dynamic equilibrium in favor of cells and tissues for this is to maintain a eukaryotic cell environment stable and self-updating of an effective way to achieve, but also its adaptation to environmental change as an important defense mechanism, but the low dose UVA irradiation adaptive response induced with autocorrelation is still reported late bite between. This observation of low dose UVA-induced adaptive response of human skin fibroblasts into autophagy, and to explore its possible role in the induction of adaptive response of cultured human skin fibroblasts fibroblasts, to clarify for the full low-dose UVA-induced human skin cells Adaptability the reaction mechanism to provide more experimental evidence.Methods1. The effect of low doses of UVA-induced human dermal fibroblasts appear adaptive response autophagy morphology and changes in the number, shape and contrast differences autophagy number of different groups of cells.2. Adoption of detection of autophagy-related protein LC3, Beclinl to explore low-dose UVA induced skin into the possible mechanism of adaptive response of fibroblasts in autophagy.3. To investigate the possible role of autophagy in cultured human skin fibroblasts induced adaptive response of fibroblasts.Methods1. Take 18-year-old healthy male penis after circumcision the foreskin (from the Guangzhou Military Command General Hospital Department of Urology outpatient operating room) in cultured human skin fibroblasts using tissue digestion.2. Group: False irradiation group:normal cultured cells after irradiation with the same treatment group were sham-irradiated placed under irradiation device; Low dose irradiation group:(1) low dose irradiation of group A:a single dose per day were 7.2 J/cm2 2、14.4 J/cm2、21.6 J/cm2、28.8 J/cm2、36 J/cm2 was irradiated; (2) low dose irradiation of group B:a daily dose of 7.2 J/cm2 irradiation, the cumulative dose was 7.2 J/cm2、14.4 J/cm2、21.6J/cm2、28.8 J/cm2、36 J/cm2. High-dose radiation group:(1) high-dose irradiation of group A:a day in a single dose was 7.2 J/cm2、14.4 J/cm2、21.6 J/cm2、28.8 J/cm2、36 J/cm2 irradiation, followed by 86.4 J/cm2 UVA irradiated cells. (2) high-dose radiation of group B:After the first cumulative doses of 7.2 J/cm2、14.4 J/cm2、21.6 J/cm2、28.8 J/cm2、36 J/cm2 low-dose UVA irradiation pre daily, then 86.4 J/cm2 UVA irradiated cells.4. Statistical Methods:GraphPad Prism 5 software for statistical analysis, all data are mean±standard deviation (x±s). After the homogeneity of variance test, two-factor analysis of variance, with P＜0.05 was considered statistically significant.Results1. tissue digestion cultured human skin fibroblasts within 24h adherent, at the beginning the majority of adherent cells were round, with prolonged adherence to gradually stretch fusiform or polygon.5-6 days for passaging, covered about 5 days after passage. Take the first 3-10 generations skin fibroblasts experimental study.2. Using laser confocal microscope, false irradiation group cell autophagy positive expression rate in 1 d、2 d、3d、4 d、5 d were 29.360±2.020、 35.850±1.513、56.100±1.365、76.000±1.579、31.440±1.662, false irradiation in the way of cell autophagy body positive expression rate of no significant difference(P＞0.05);Low dose irradiation cell autophagy body positive expression rate of group A in 1 d、2 d、3 d、4 d、5 d were 13.677±1.528、 37.333±1.528、57.333±2.082、79.333±1.528、36.330±1.528, compared with false exposure group had significant difference (P＜0.01); Low dose irradiation autophagy body positive expression rate of group B cells in the 1 d＞2 d, 3d、4 d、5 d were 13.677±1.284、32.000±2.000、46.677±1.528、57.000± 2.000、30.000±1.000, compared with false exposure group had significant difference (P＜0.01); low dose irradiation autophagy body positive expression rate of group A and low dose irradiation of group B cells are increased with the increase of irradiation dose, but after 28.8 J/cm2 cell autophagy appears positive expression rate reduced, low dose irradiation cell autophagy body positive expression rate of group A is higher than low dose irradiation group B, low dose irradiation group of A compared with low dose irradiation group of B there were significant differences (P＜0.01). High dose irradiation cell autophagy body positive expression rate of group A in 1 d、2 d、3 d、4 d、 5 d were 29.360±2.020、80.130±3.096、45.140±1.845、19.630±0.772、7.537 ±0.463, compared with false exposure group had significant difference (P＜0.01); High dose irradiation autophagy body positive expression rate of group B cells in the 1d、2 d、3 d、4 d、5 d were 29.360±2.020、35.850 ±1.513、56.100±1.365、76.000±1.579、31.440±1.662, compared with false exposure group had significant difference (P＜0.01); High dose irradiation group of A in 14.4 J/cm2 after exposure to 86.4 J/cm2, cell autophagy positive expression rate reduce, high dose irradiation group of B cell autophagy body positive expression rate in 28.8 J/cm2 reduce after exposure to 86.4 J/cm2, high dose irradiation with high dose irradiation group of B compared with group of A significant difference (P＜0.01).3. The positive expression rate and cell autophagy body consistent test results, Beclinl, LC3 expression in low dose irradiation group of A and low dose irradiation group of B were increased with the increase of irradiation dose, but after 28.8 J/cm2 Beclinl, LC3 expression levels drop, low dose irradiation Beclinl, LC3 expression levels of group A were higher than low dose irradiation group of B, low dose irradiation group of A compared with low dose irradiation group of B there were significant differences (P＜0.01); High dose irradiation compared with high dose irradiation group of B, group of A high dose irradiation Beclinl, LC3 expression peak value and then quickly cut quickly, and high dose irradiation group of B Beclinl, LC3 expression can still slowly increases with dose accumulation peak value didn’t appear. High dose irradiation group of A compared with high dose irradiation group of B there were significant differences (P<0.01).Conclusion1. Human skin fibroblasts induced by low dose UVA irradiation in adaptive response at the same time can cause the change of human skin fibroblasts autophagy, the rule of the change is closely related to the dose, the related regulation and LC3, Beclinl expression.2. The results showed that autophagy in human skin fibroblasts induced by low dose UVA irradiation in adaptive response may play an important role. |
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