| Background The increased pigment disease of skin like chloasma is common in clinic. The current treatment of chloasma is limited and has poor curative effects, the recurrence rate is high. The factors involved in skin pigmentation are very complex, the pathogenesis is unclear too. In order to find better treatment, improve curative effects and reduce the recurrence rate, we try to find out the etiology and pathogenesis of chloasma. Recent studies found that the estrogen receptor was expressed higher in the patients with chloasma. The UVB irradiation can promote the melanin cell proliferation and division, increase the melanin synthesis, and can promote the melanin transport by enhancing the exocytosis of melanocytes. The objective of this project is to establish a suitable model in vitro to study the role of UV and estrogen in pigmentation diseases of skin. Objective To investigate the influence of different levels of estradiol and ultraviolet B(UVB) radiation on the melanosome metabolism of epidermis by establishing a keratinocytes and melanocytes co-culture system in vitro and guinea pig animal model, and explore the effects of estrogen and UVB in the pathogenesis of chloasma. Methods 1. The original generation of melanocyte were cultured by using fresh foreskin tissue, then the immortalized keratinocytes cell lines(Ha Ca T cells) were recovered from the liquid nitrogen tank. After the steps of cell medium exchange, cell passage and etc, the co-culture cells got good biological activity and were in logarithmic growth phase. The inverted microscope and cell immunohistochemical techniques were used to observe the growth condition and morphology of the co-culture system in vitro. The co-cultured cells were treated with different concentrations of estradiol combined with UVB irradiation. The control group was set in the experiment. The groups were divided into DMSO control group, UVB irradiation group, estradiol-treated group and estradiol-pretreated combined with UVB irradiation group(estradiol + UVB group). The estradiol concentration was set to 10-5mol/L, 10- 6mol/L and 10-7mol/L. The UVB irradiation dose was unified 20 m J / cm2. Use MDC staining method to detect the autophagy levels of Ha Ca T cells in co-culture system after treatment. 2. Healthy suit guinea pigs were selected in this study. The skin of each guinea pig’s back was divided into solvent control group, UVB irradiation group, estradiol treatment group and estradiol pertreatment combined with UVB irradiation group(estradiol + UVB group). The concentrations of estradiol were set to 3.67×10-5、3.67×10-4、3.67×10-3 mol/L. The UVB exposure dose was unified as 500 m J/cm2. The brown fur on the back of the guinea pig were disposed with a certain concentration of estradiol and UVB radiation, then the naked eye pictures and skin color measurements before and after disposing were compared. Transmission electron microscopy and in vivo confocal laser scanning microscopy(referred to as skin CT) were used to observe the effects of estradiol and UVB in skin pigmention on the back fur of the guinea pig. Thus the probable role of estradiol and UVB in melanin compound and degradation were observed. Results 1. Human melanocytes and Ha Ca T cells had a good cell activity when cultured in vitro alone. In the established co-culture system, the proportion of Ha Ca T cells and melanocytes was increased significantly with time prolong(P<0.05). MDC staining showed that: after a certain concentration of estradiol treatment combined with UVB irradiation, the percentage of positive cells of autophagy did not change significantly, compared with the control group(P>0.05). There was no statistical difference. 2. In the animal experiments, the pigmentation of guinea pig skin were deepened after a certain concentration of estradiol treatment, and the pigmentation was increased gradually with the increasing of estradiol concentration(P<0.05). The pigmentation of guinea-pig skin were also deepened significantly in UVB irradiation group compared with non-UVB irradiation group(P<0.05). The autophagy of epidermal keratinocytes of guinea pigs were increased after UVB irradiation, but were decreased after estradiol treatment. Conclusion 1. In the human melanocytes and Ha Ca T cell co-culture system, a certain concentration of estradiol and UVB irradiation treatment fail to enhance the autophagy activity in Ha Ca T cells. 2. In the animal model experiments, estradiol treatment and UVB irradiation can promote pigmentation of guinea pig skin and the mechanism may be related to the promotion of melanosome synthesis. The degradation of abnormal melanosome may also be involved in the formation of pigmentation. |
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