| Background and objectiveUltraviolet phototherapy (UVA and UVB) is one of the most important methods of skin diseases treatment. Epidermis is the main location of ultraviolet B action, and wavelengths of 311-313nm narrow-band UVB(NB-UVB) is the most effective component of ultraviolet B. In recent years, NB-UVB phototherapy had been widely used for the clinical treatment of various skin diseases such as vitiligo, psoriasis, atopic dermatitis and so on and had achieved better therapeutic efficacy. Both melanocytes and keratinocytes of epidermis are main target cells of NB-UVB.Our studies focused on the influence of NB-UVB radiation on cultured epidermal cells in two sections to investigate: 1. The effect of different NB-UVB radiation dose and action time on the morphology, proliferation activity and synthesis of melanin of melanocytes from both normal appearance skin and the edge of lesion that were cultured in patients with vitiligo, setting cultured melanocytes from normal healthy individuals donors as the control group, and to determine the time point of maximal cell proliferation rate and synthesis of melanin, in order to guide clinical treatment setting optimal NB-UVB radiation interval time. 2. The correlation of effect of NB-UVB radiation on cultured immortalized human keratinocyte cell line -HaCaT cells apoptotic morphology, proliferation activity and the rate of apoptosis with NB-UVB radiation dose and detection time, and to investigate the mechanism of action of NB-UVB for treatment of psoriasis and the optimal NB-UVB radiation treatment frequency. Methods1. Melanocytes from normal appearance skin, the edge of lesion of patients with vitiligo and the normal healthy individuals donors were cultured. Monoclonal antibodies, such as HMB-45, were used to identify cultured melanocytes. Effect of NB-UVB radiation on morphology of melanocytes was observed by invert microscope. MTT method and NaOH assay were applied to detect the effects of NB-UVB radiation dose (20 -100 mJ/cm~2) and action time (48h, 72h and 96h after radiation) on cell proliferation and synthesis of melanin.2. HaCaT cells were irradiated with NB-UVB in dose of 20, 40, 60, 80, 100 mJ/ cm~2, then we observed the damage effects of NB-UVB radiation on cell morphology and proliferation activity detected on the time of 24h, 48h and 72h after radiation. Cell apoptosis was detected by flow cytometry. Results1. NB-UVB radiation resulted in cellular swelling, cytodendrites shortening and thickening, and decrease in cellular adhesiveness.NB-UVB radiation enhanced the capacity of melanogenesis in a dose-dependent manner at 48h after radiation. Radiation dose≥60mJ/cm~2 inhibited proliferation of melanocytes from normal appearance skin of patients with vitiligo and the normal healthy individuals donors, while the dose≥40mJ/cm~2 produced inhibitory action of melanocytes from the edge of lesion of patients with vitiligo; NB-UVB radiation promoted cell proliferation and melanogenesis at 72h and 96h after radiation, the effect on melanocytes from normal appearance skin of patients with vitiligo and the normal healthy individuals donors was more obvious, and 72h after NB-UVB radiation was the time point of maximal cell proliferation rate and synthesis of melanin.2. NB-UVB radiation could severely damage HaCaT cells by inducing cell shrinkage and fragment increases. Treatment with NB-UVB increased the number of HaCaT cells possessing apoptotic morphologic characteristics and decreased the cell viability in a dose- and time-dependent manner within 48h after radiation, then the damage effects got slight . At the time of 24h after radiation, dosage of 20-100 mJ/cm~2 NB-UVB radiation could induce HaCaT cells apoptosis in a positive dose-dependent manner. Conclusions1. Melanocytes from the edge of lesion of patients with vitiligo were quite sensitive to NB-UVB radiation, the degree of effect of photodamage was serious, and the function of synthesis of melanin also existed abnormality.2. Melanocytes from normal appearance skin of patients with vulgaris vitiligo had the similar biological characterictics with melanocytes from the normal healthy individuals donors.3. The greatest effect on the cultured melanocytes of vitiligo occurred at 72h after NB-UVB radiation, which indicated that patients with vitiligo could achieve better therapeutic efficacy by radiating NB-UVB twice a week.4. NB-UVB radiation could decrease the proliferation viability of HaCaT cells in a dose-dependent manner, and the most obvious damage effects of morphology changes and proliferation inhibition occurred at the time of 48h after NB-UVB radiation , which indicated that patients with psoriasis could achieve better therapeutic efficacy by radiating NB-UVB thrice a week.5. NB-UVB radiation could induce HaCaT cells apoptosis in a dose-dependent manner.6. NB-UVB radiation produced a local marked effect on keratinocytes for treatment of psoriasis partly by inducing cell apoptosis and suppress cell proliferation ability to achieve the regulation of keratinocytes excessive proliferation. |
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