The Function Of Microvesicles On UVA/UVB-induced Bystander Effects

Background and ObjectiveSolar ultraviolet radiation induced skin disease has been a concern point of interest disease. Studies have indicated that besides to induce the direct damage of. the radiation cells, ultraviolet radiation also through the ultraviolet radiation induced bystander effects to cause the collateral damage of adjacent cells.In recent years, the study on the bystander effects of ultraviolet radiation has covered a variety of light damage models. Researchers have found that different wavelengths of ultraviolet radiation can produce varieties of bystander effects. In addition, different doses of ultraviolet radiation can induce different intensity of the bystander effects. All the evidence shows that the bystander effects of ultraviolet radiation may play an important role in light induced skin damage, photocarcinogenesis, and other important pathological process. The bystander effects of previous research were focused on the ion radiation, although as a non-ionizing radiation, the evidence of bystander effects induced by ultraviolet radiation has established well. While the mechanism of biochemical and molecular mechanism of the bystander effect has just begun, this research attracts many scholars’ attention.In recent years, the function of the microvesicles and the information they carried are hot research topic in biology. Research reports cells exposure to ionizing radiation can excrete microvesicles, which are cystic structures resource of budding and then fall off from membrane, carried protein, mRNA, miRNA and DNA. Microvesicles participate in intercellular information transmission, and mediated bystander effects. The aim of this study is to observe the production of microvesicles in human skin fibroblasts, and the effect of cell morphology, proliferation activity, oxidative damage and apoptosis of human skin fibroblasts (HSF) in acute UVA/UVB radiation.Methods1. Function and identification of UVA/UVB-induced microvesicles in human skin fibroblastsUsing different doses of UVA/UVB irradiation on human skin fibroblasts, establish the cell models of acute light damage. In this study, the radiation doses of UVA were 10J/cm2 and 20J/cm2, and the radiation doses of UVB were 30mJ/cm2 and 60mJ/cm2. The cells were cultured with serum which not contain any microvesicles then irradiated by UVA/UVB. After irradiation 0.5h,24h,48h,72h, microvesicles were separated and extracted from the cell culture supernatant. Subsequently, use transmission electron microscopy (TEM) and light scattering technique for analysis the size and content of microvesicles, BCA protein quantitative method for determination of microvesicles protein in the culture supernatant, Western blotting for detection of microvesicles surface proteins.2. Effects of acute UVA/UVB irradiation induced microvesicles on cell morphology, proliferation activity, oxidative damage and apoptosis in human skin fibroblastsHSF were acute irradiated by UVA (20J/cm2) and UVB (60mJ/cm2), then collected the microvesicles in the cell culture supernatant after 24h. The microvesicles were incubated with HSF after labeled with PKH26 dye, and then observed the uptake rate of microvesicles. Bystander cells morphology was observed under inverted microscope, cell counting CCK-8 assay and EDU staining method in the detection of the bystander cells proliferation rate. Reactive oxygen species (ROS) kit for the detection of bystander cells oxidative damage, mitochondrial membrane potential detection kit for the detection of early apoptosis, apoptosis rate was detected by membrane associated protein V (annexin V)/propidium iodide (PI) double staining flow cytometry.Result1. Function and identification of UVA/UVB-induced microvesicles in human skin fibroblastsUVA/UVB induced microvesicles was significantly higher than normal HSF. TEM showed that microvesicles are kinds of microcapsule with intact membrane, round or oval, with low electron density components; light scattering techniques suggest that the diameter of microvesicles excreted by ultraviolet radiation damage almost gathered in the 150-300nm, while the microvesicles resource from normal HSF were 120-200nm, and the amount of microvesicles after ultraviolet radiation higher than normal secretion of HSF; BC A protein quantitative results illustrated that the amount of UV induced microvesicles showed a secretion peak in between 24h-48h, while microvesicles derived from the normal cells have a relatively constant in different time points; western blotting results suggested that the UV induced microvesicles give positive expression of surface protein Alix, TSG101 and CD9, and the expression was significant in Alix.2. Effects of acute UVA/UVB irradiation induced microvesicles on cell morphology, proliferation activity, oxidative damage and apoptosis in human skin fibroblastsMicrovesicles, which labeled by PKH26 fluorescent, can be uptake by the bystander HSF after adding the culture medium for 30min. Follow with the incubation in 48h, the nucleus cells gathered around microvesicles were increased gradually. The uptake rate of ultraviolet radiation induced microvesicles was faster than normal HSF induced microvesicles. After the cells were incubated, under an inverted microscope appear more cell debris, cell volume increase and hyperextension; CCK-8 showed bystander HSF proliferation become slow; EDU staining showed cell cycle arrest, S phase cells in bystander cell increased; ROS results suggested that the strength of active oxygen fluorescent bystander cells was higher than that of the non-irradiation group; mitochondrial membrane potential suggest that bystander HSF appears to have more cell debris and more green fluorescence, which indicating early apoptosis; AV/PI staining showed that bystander HSF apoptosis rate increased; while the cellular oxidative damage and apoptosis rate can be partially reversed by NAC, which is an antioxidant.ConclusionAcute UVA/UVB radiation can induce skin fibroblasts to release microvesicles, which can be uptake by bystander cells, lead to the changes in cell morphology, reduce the cell proliferation rate, cell cycle arrest, stimulate oxidative damage and apoptosis of the cells.