The Effect Of Ultraviolet B Radiation On The5-methylcytosine And5-hydroxymethylcytosine Levels Of Human Fibroblasts

Objectives:1. Observe the effect of Ultraviolet B radiation on the proliferation ability of cultured human fibroblasts.2. Observe the effect of Ultraviolet B radiation on the5-methylcytosine (5-mC) and5-hydroxymethylcytosine (5-hmC) level of cultured human fibroblasts.Methods:1. Divide primary cultured fibroblasts into two groups: control group and treatment group (accumulative dosage of50mJ/cm2UVB irradiation);2. Detect the fibroblasts proliferation activity by MTT assay;3. Observe morphological changes of two groups in HE stained fibroblasts;4. Detect5-hmC and5-mC expression of fibroblasts by immunohistochemical ABC method;5. Detect5-hmC and5-mC expression of fibroblasts by immunofluorescence.Results:1. Morphological changes were observed by HE staining, control group fibroblasts arranged in order, the swirled shape or palisade, fusiform cell morphology, plump cytoplasm, the cell membrane, visible mitotic;cells become large, flat, mostly manifested polygonal loose and transparent cytoplasm, boundaries become unclear nucleus particles increased mitotic rare in50mJ UVB irradiation;2. MTT test results of the absorbance value of the UVB treated cells slightly increased with time (#24,48,72,96,120hours5time points), while the absorbance value of the blank control group cells was significantly increased with time;the control group in72h,96h,120h at each time point absorbance values were significantly higher than the UVB irradiation group (P<0.05);3.The immunohistochemical ABC method results showed that5-hmC expression of the control group was significantly lower than the treatment group, while the5-mC expression was significantly higher than the treatment group (P<0.05);4. Immunofluorescence results were the same with the immunohistochemistry ABC method:5-hmC expression of the control group was significantly lower than the treatment group, while the5-mC expression was significantly higher than the treatment group (P <0.05). Conclusion:1. Sub-toxic dose (50mJ/cm2) of the UVB radiation significantly inhibited cell proliferation of fibroblasts cultured in vitro;2. UVB radiation decrease the5-mC expression level of fibroblasts cultured in vitro, meanwhile increase the5-hmC expression level.