The Effect Of Heat Treatment On The Activity Of Cultured Normal Human Melanocytes Irradiated By UVB

ObjectiveVitiligo is the most common skin disease which is characterized by local orgeneralized depigmentation of the skin and mucous membranes. It usuallypresents in childhood or young adulthood, with a prevalence of approximately0.1～2%in the world population independent from race and gender. The lesionsmay occur elsewhere, and the pathologic hallmark is the reduction ordisappearance of epidermal melanocytes. The etiology of vitiligo is stillunknown, and several factors may contribute to the pathogenesis of vitiligo,including genetic predisposition, autoimmune, oxidative stress, neural theories.The current treatments of the disease include drug, phototherapy, surgery.Phototherapy, especially UVB radiation at a wavelength of311～313nm, whichis more effective and safer, has been used increasingly for the management ofvitiligo.Recently, more and more evidences indicated that heat treatment, which is similar to UV，can increase the tyrosinase activity and melanin content in MC.Furthermore, the number of the active MC was increased in the KC-MCco-culture system after the treatment with heat. The main solar energymaterial—heat (infrared ray) may also increase the tolerance of the skin cells toUV. These results suggest that the combination therapy of heat and UVB willprobably become an important treatment of vitiligo. This study investigated theeffect of heat treatment on the viability frequency, tyrosinase activity, melaninsynthesis and cell cycle of cultured normal human melanocytes irradiated byUVB, and expected to provide a theoretical basis for the combination therapy ofheat and UVB in the depigmentation skin disease.Methods1. The cultured normal human melanocytes were irradiated with20、30、50、70、90、120、180mJ/cm~2dosages of NB-UVB, MTT were used to detect theirproliferation and cellular activity, the best dosage of UVB will be chosen andapplied in the next step of the experiment.2. Melanocytes were treated with heat (42℃,1h), UVB radiation(50mj/cm~2)or both of them(first50mj/cm~2,then42℃,1h) for3days. After another24hoursculture, the proliferation and cellular activity of melanocytes were detected byMTT, tyrosinase activity and melanin content were detected by DOPA oxidaseand NaOH methods. The responses of cell cycle were examined by immunefluorescence technique.Results1. After irradiated with20、30、50、70、90、120、180mJ/cm~2dosages ofNB-UVB, the cell viability frequency of melanocytes decreased in a dose dependent manner. The optimum irradiation dose was at50mj/cm~2.2. Compared with the control cells after the different treatments, theviability frequency of melanocytes in UVB group decreased7.1%（P<0.05）,heat group and the united group increased25.3%and58.1%（P<0.05）. Thecombination of heat and UVB has a synergistic effect.3. Compared with the control cells after the different treatments, thetyrosinase activity and melanin content was significantly increased. The growthrate of tyrosinase activity in UVB group,heat group and the united group were3.8%、11.5%and31.9%（P<0.05）. The growth rate of melanin concentrationin UVB group,heat group and the united group were17.5%,31.6%and67.3%（P<0.05）. The combination of heat and UVB has a synergistic effect.4.Compared with the control cells, the percentage of cells in G1-phase inUVB group, heat group and the united group decreased24.61%、13.16%and32.02%（P<0.05）, the percentage of cells in S-phase in three groups increased15.02%、8.58%and17.14%（p<0.05）, the percentage of cells in G2-phase inthree groups increased15.02%、8.58%and17.14%（p<0.05）. The combinationof heat and UVB has a synergistic effect.Conclusions1. Heat treatment (42°C,1h) could promote proliferation of melanocytes.The combination of heat and UVB has a synergistic effect.2. Heat treatment (42°C,1h) and UVB radiation (50mj/cm~2) cansignificantly increase tyrosinase activity and melanin content of cultured normalhuman melanocytes, and the united group had a synergistic effect.3. Heat treatment (42°C,1h) and UVB radiation (50mj/cm~2) can promotecell cycle arrest in S phase and G2phase, and reduce the number of melanocytes in the G1phase. The combination of heat and UVB has a synergistic effect.