Effect Of Heat Stress On Growth And Melanin Synthesis Of Cultured Normal Human Melanocytes

ObjectiveThe color of human skin was maintained mainly by melanin synthesis of epidermis melanocytes and melanin transfer to surrounding keratinocytes. If the function of melanin synthesis and transfer of melanocytes are abnormal, it will lead to a variety of skin pigmented disease(such as: vitiligo,melasma etc.). Therefore it has very important clinical significance for the prevention and treatment of skin pigmented disease to study the influential factor and mechanisms of melanin synthesis and transfer of melanocytes.Sun exposure can cause the color of the skin to be tan, previous studies have thought that these changes are mainly related to ultraviolet (UV), particularly ultraviolet B (UVB), but more and more evidences indicated that the main solar energy material—heat (infrared ray) and UV have similar effects on the skin, and heat may also increase the tolerance of the skin cells to UV. The end of last century, Nakazawa[1] studied human epidermal MC response to the two kinds of exogenous stimulation(heat and UVB). The study found that heat-treated or UVB-treated melanocytes showed an increased dendricity and a larger cell body compared with nontreated melanocytes, the tyrosinase activity and melanin content per cell was increased ,but MC had a low survival than nontreated MC after the treatments with heat(42℃,60min)or UVB(20mJ/cm~2). At the same time, the study also found that the number of the active MC was increased in the KC-MC co-culture system and tissue-engineered skin after the treatments with heat or UVB. So the author considered that heat shares significant biologic activities with UVB in melanocyte functions. But then Kim[2,3]raised an opposite view, their investigation demonstrate that heat treatment reduces melanin production of mouse MC cell line (Mel-Ab) and human epidermal MC in a temperature-dependent manner .To further clarify the regulation action of heat on physiological functions of human epidermal MC, cultured normal human epidermal MC were utilized to study the effects of heat stress on proliferation activity, cell morphology, tyrosinase activity and melanin synthesis of MC in this study, and the fuction of heat shock protein 72 (HSP72) and p53 was initially investigated from the levels of the proteins and genes in the heat stress induced activity changes of MC.Methods1.To obtain the foreskin of normal human after sterile circumcision and harveste melanocytes according to epidermal cell culture methods.Basic fibroblast growth factor(bFGF) was utilized to establish the culture of normal human melanocytes. Masson-Fontana staining method was employed to determine germ-line of cells. 2.After the different doses of heat stress (39℃、41℃、42℃、43℃、45℃) were applied in cultured normal human melanocytes, the cell viability was measured by tetrazolium blue (MTT) colorimetric assay. The best temperature will be chosen and applied in the next step of the experiment .3.Cell morphology, tyrosinase activity and melanin content of the optimum heat stress (42℃) on the melanocytes were compared with the normal control temperature (37℃). Protein and gene expression of HSP72 and p53 under heat stress condition were compared with the control group by Western-bloting and RT-PCR.4.All of date analysis were done using the SPSS 13.0 statistical software package .Results1. Heat treatment changed the configuration of the melanocytes into an activated form with an increased number of dendrites and enlarged cell body compared with the control. These morphologic changes were observed after approximately 24h of the last treatment with heat. The results of electron microscopy show that the density of melanin granules was higher in treatment group than nontreatment and the envelope of melanosome was intact in both of groups. No significant damage of the organelles was found.2. Melanin content(0.152±0.005) in vitro cultured normal human melanocytes which were treated by heat (42℃for 1 h per day) for 3 days was significantly higher than nontreated melanocytes(0.086±0.005), the growth rate of melanin content is 78%( t=24.81,P<0.01). Under the same conditions, tyrosinase activity of melanocytes in the experimental group(0.193±0.004)was significantly higher than the activity of melanocytes in control group(0.142±0.011), the growth rate of tyrosinase activity is 36.4%( t=9.34,P<0.01).3. The expression levels of HSP72 and p53 proteins in vitro cultured normal human melanocytes which were treated by heat (42℃for 1 h per day) for 3 days were obviously increased compared with nontreated normal human melanocytes after 6h、12h and 24h. Under the same conditions, the expression levels of HSP72 and p53 gene were also obviously increased after 12h.ConclusionsThe experiment results show that cell appearance of vitro cultured normal human melanocytes which were treated by heat (42℃for 1 h per day) for 3 days was significantly better than nontreated melanocytes, and the biological activity of melanocytes in the experimental group were also significantly enhanced compared with normal control group. This indicates that heat stress may play a positive regulatory role on biological functions of normal human melanocytes in vitro. At the same time, we found that HSP72 and p53 protein in the experimental group express more than the normal control group. This prompt that both kinds of proteins may play an important role in regulating biological activity of melanocytes. These provide us an important theoretical basis for the further study of the mechanism of heat stress on melanocytes, and may opened up a new way for the prevention and treatment of pigmented skin.