Dissertation
Dissertation > Medicine, health > Dermatology and Venereology > Dermatology

Action of Ultraviolet Radiation on Proliferation of Human Melanocytes and Melanin Synthesis

Author WangLei
Tutor WangBaoZuo
School Peking Union Medical College , China
Course Dermatology and Venereology
Keywords Melanocytes Radiation dose Melanogenesis The number of cells Ultraviolet radiation Medical Melanin synthesis Pheomelanin Master's degree thesis Priority melanocytes
CLC R751
Type Master's thesis
Year 2007
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Action of Ultraviolet Radiation on Proliferation of Human Melanocytes and Melanin SynthesisThe sunlight is divided into three categories (UVC-200 to 280nm, UVB-280 to 320nm and UVA 320 to 340nm) based on the wavelength. Among them UVB and UVA are closely related with the number of melanocytes and melanin’s synthesis. Ultraviolet radiation (UV) induces a variety of responses in the skin, including tanning and inflammation, and may also act as a carcinogen.Recent studies have proved that UVA and UVB play critical roles in the proliferation of melanocytes and pigment formation. UVB has stronger effects on melanocytes in comparing with UVA, it can increase the proliferation of the melanocytes and induce apoptosis of the cells on the other hand. UVB can also promote the synthesis of melanin and delayed tanning (DT) after sun-exposure. In the contrast, UVA has little effect on melanocytes and melanin synthesis.The present study was conducted to evaluate the direct effect of UVA and UVB irradiation on melanocytes in vitro. Melanocytes isolated from foreskins of different healthy adult men in the laser clinic of PUMC hospital and were cultured in M-254 medium containing melanocytes growth supplement and antibiotics. In this study,melanocytes between passage 3 and 5 were used.1. Detection of melanocyte proliferation against UVA and UVB irradiation. We applied an MTT assay to determine the maximum doses of UVA and UVB irradiation. Briefly, melanocytes grown in 96-well plates at 70-80% confluence were divided into three groups. One group of the cells was exposed to different doses of UVB and incubated in complete medium for further 48 hours. Another group of melanocytes was exposed to different doses of UVA and incubated in complete medium for further 48 hours. Each dose of UVA and UVB was repeated in 8 wells. The third group grown in complete medium without UV irradiation was used as a normal control. In each well 20μL of MTT solution(5g/L) was added and the culture was continued for a further 4 hours. The reaction was stopped by adding 100μL DMSO and the absorbance of each well was measured at 570nm with a Automated EIA analyzer.2. Detection of tyrosinase activity against UVA and UVB irradiation: An assay was applied to measure the activity of UVA and UVB irradiation on the cells. In brief, melanocytes at 70-80% confluence were incubated in 3.5mm dishes. The dishes were divided into three groups. One group of melanocytes was exposed to different doses of UVB and incubated in complete medium for further 48 hours. Another group of melanocytes was exposed to different doses of UVA and incubated in complete medium for further 48 hours (each dish for a dose of UVB or UVA). The third group grown in complete medium without UV irradiation was used as a normal control. In each dish the medium was drained and Trixton X-100 solution(0.1%) was added and vibrated for 15 min. The solution was transferred into an Eppenddorf. L-DOPA (1%)was added until the volume of solution reached 1 ml. The cell supernatant was transferred into 96-well plates for 100μL per well. The absorbance of each well was measured at 490nm with a Automated EIA analyzer.3. Detection of melanin content against UVA and UVB irradiation: the methods of grouping and irradiation were the same as mentioned above. In each dish the medium was drained and NaOH(1 M) was added and vibrated for 10 min. The solution was transferred into an Eppenddorf. NaOH (1M) was added until the volume of solution reached 1 ml. The cell supematant was transferred into 96-well plates for 100μL per well. The absorbance of each well was measured at 450nm with an Automated EIA analyzer.Results:1.Proliferation of melanocytes after UV irradiation: the maximum dose of UVA irradiation was 72mJ/cm~2 and that for UVB was 14.4 mJ/cm~2. When the dose of UVA increased higher than 72 mJ/cm~2 , the proliferation of melanocytes slightly decreased but still higher than that of the normal control group. On the other hand, as the dose of UVB exceeded 14.4 mJ/cm~2, the proliferation of melanocytes distinctly decreased, the higher the dose, the less the proliferation.2. Tyrosinasc activity of the cells aider UV irradiation: the maximum dose of UVA irradiation was 100 mJ/cm~2, which was corresponding with the proliferation of mclanocytcs. As the dose of UVA increased, the tyrosinase activity decreased. The higher the dose, the less the tyrosinase activity.For UVB, on the contrast, the maximum dose is 10mJ/cm~2, corresponding with the proliferation of the cells, either. When the dose of UVB was higher than 10mJ/cm~2, the tyrosinase activity was still higher than that of the control group.3. Melanin synthesis after UV irradiation: the maximum doses of UVA irradiation was 200 mJ/cm~2. When the dose increased, the contents of melanin was still higher than that of the control group. For UVB, the maximum dose was between 10 and 20mJ/cm~2, corresponding with the dose of the maximum mclanocytc proliferation. When the dose was higher than 20mJ/cm~2,the melanin content slightly decreased but still higher than that of the control group.Conclusion:1. UVA slightly promoted the proliferation and melanin synthesis of mclanocytes, barely induce the apoptosis of the cells in vitro.2. UVB significantly promoted the proliferation of melanocytes even at low dose and strongly induced the apoptosis of the cells. It also significantly induced the activity of tyrosinasc of the cells to produce melanin.

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