| BackgroundPure white and tender skin is taken for fair for the Asian ethnic. Pigmentation or stains appearing on the skin greatly affect their appearance. There are many types of acquired pigmentation disorders, for example melasma, freckles, sunburn plaques, senile plaques, post-traumatic pigmentation, pigmentation after surgery and so on.There are many reasons for pigmentation, and its etiological factor is very complicated. Any one affecting the course of pigmentation may lead to this disease. The process includes:①melanosome assembly and melanin synthesis in melanocytes;②melanosome transfer from nuclear surrounds to dendritic remote;③melanosome transferred to neighboring keratinocytes;④melanosome re-distribution and degradation in keratinocytes. The major reason could be divided into genetic factors, physical factors (such as ultraviolet light, sunlight exposure, etc.), chemical factors (heavy metals such as arsenic, bismuth, silver, mercury and other incentives), endocrine factors (pregnancy, pituitary, thyroid, adrenal gland, gonadal function disorders, such as change of progesterone and estrogen levels), inflammatory factors and metabolic factors, and so on.Estrogen is a steroidal hormones mainly produced by the follicle and corpus luteum. The main role is to promote the development of the reproductive system, to stimulate the female secondary sexual characteristics, and affect metabolism. Studies show that estrogen is closely related with breast disease, reproductive system diseases, cardio-cerebral blood vessels, bone and joint diseases.Clinically we found that pregnancy, oral contraceptives, chronic diseases, cosmetics may lead to skin pigmentation, a typical example such as melasma. The hormone levels'change such as the increase of estrogen, progesterone hormone and MSH level blood in vivo of pregnant women may result in the increase of activity of melanocytes, and increase melanin synthesis. The pigmentation often appears in the pregnancy early, middle period, and gradually become obvious at last. And after delivery melasma will continue its existence for some patients. Long term oral contraceptives for women, melasma will be induced like as the pregnancy due to the change of hormone levels in vivo. Generally pigmentation will appear after taking medication for 20 months and gradually disappear until discontinuation drug. Melasma often appear in some chronic diseases such as liver disease, chronic alcoholism, tuberculosis, visceral tumors, thyroid disease and some autoimmune diseases, in particular female genital diseases and irregular menstruation, dysmenorrhea, uterine attachment inflammation, infertility. This may be related to the ovary, pituitary, thyroid and other endocrine hormone levels disorders. Estrogen may play an important role in the incidence of the above diseases, but the relationship between estrogen and pigmentation is rarely study.In addition, melasma often appear in Asia, Latin America ethnic, and rarely occur in the white race. Therefore the role of estrogen in the pigmentation disease is rarely researched.Human melanocytes mainly distribute in the human epidermis, dermis, hair follicle and eye choroid, etc. Melanocytes in the epidermal basal layer and hair follicles have the capacity of melanin secretion and synthesis. There are 100-2000 million melanocytes in the basal layer of the human epidermis, average about 1560 cells in per mm2. They distribute in the body surface symmetrically, but the depths vary due to different sites, generally more in head and skin folds, less in abdominal and back.Maedak and other scholars have incubated normal human melanocytes with pituitary hormones, promoting melanoma cell hormones, FSH and ovarian hormones for estradiol (E2), estriol (E3), progesterone (P). After incubating for 2 days melanocytes became lager and took on dendritic-like appearances. They found in the trials that pituitary hormone can increase the activity of tyrosinase and tyrosinase-related protein-1, but ovarian hormone can only increase the activity of tyrosinase-related protein-1, without increasing the activity of tyrosinase in the same experimental conditions. This suggested that skin pigmentation caused by pituitary and ovarian hormones may be resulted by stimulating epidermal melanin generation in melanocytes, and E2 and P play an important role in the pathogenesis of melasma.Some scholars have found estrogen and progesterone hormone levels in serum do not increase, sometimes even lower than normal in melasma patients. Especially melasma appears in some men. On the other hand pigment becomes deepen only in about 50% of pregnancy patients and oral contraceptives. So it is not the increase of sex hormone level which leads to melasma. Perhaps melanocytes in this part of patients is more sensitive to hormone, also perhaps there are differences in the melanoma cell-surface sex related hormone receptors of these patients.Since the discovery of estrogen receptor (ER), reports on estrogen receptor is endless. Previous study on ER is limited to ERa. Until in 1996, a high affinity estrogen receptors ERβis isolated from the human and rat prostate tissues respectively. So researches on molecular biology of ER have entered a new stage particularly in the histological positioning of the two receptor subtypes and its relationship with gynecological endocrine. It is generally agreed that ER exists in multiple organs in vivo, and is closely related with menopausal and postmenopausal-related diseases. ERa is composited by 595 amino acids, which molecular weight is 66 KD and gene maps on chromosomes 6q25.1 zone, containing more than 140 kb of the base pair. ERβwas composited by 485 amino acids, which molecular weight of 54 KD, gene mapping on chromosome 14q22-24 areas, about 40 kb. ERa and ERβof human is very similar in the C domain and E domain, there is 97% and 59.1% homology. But there is only 17.5% and 17.9% homology in A/B domain and F domain. ERa and ERP exist in gender genital, heart, brain, bone, and kidney and so on widely. Combination with estrogen exert specific role in different organs.Estrogen binding with cell surface estrogen receptor or other receptor activates the specific second messenger signal transduction system. When there is no estrogen, ER binds with co-inhibitor, and with no transcriptional activity. When estrogen in the tissue specific binding with ER, ER dissociates with co-inhibitor, phosphorylation occurs, dimers forms, and the co-activator factor is recruited, which activates transcription, then related proteins syntheses, and estrogen-target effects is exert. At present, it is considered there is two ways for ER to regulate the gene expression. First, ligand-dependent regulation, namely, the activated androgen receptor complex combined with specific estrogen response element (ERE) in DNA sequence, which directly activated the transcription; or binding with activated protein (API) transcription factor complex to control transcription indirectly. Second, non-ligand-dependent regulation, such as epidermal growth factor (EGF) can induce ERa phosphorylation and activate the transcription.In short, the estrogen not only affects the skin hyperkeratosis, dermal fibrosis, melanin formation, but also regulates the function of skin appendages such as hair follicle, sweat glands, and sebaceous glands. So it is important in skin aging, pigment formation, hair growth, gland secretion, and skin cancer formation.The studies on the estrogen mainly concentrate on the role of the tumor, osteoporosis, and cardiovascular disease pathogenesis at present. As for estrogen and its receptor in the role of skin pigmentation have not been reported.Objective1,To investigate the histological changes of the skin with melasma and keratin 10 (CK10) expression. To study the melanin distribution in the melasma lesions and its differences with normal skin.2,To investigate the pigment characteristics and the estrogen changes in serum of female melasma patients.To investigate the distribution of estrogen receptor (3(ERβ) in the skin of of melasma patients, to understand the role of estrogen in the pathogenesis of melasma. To investigate the culture method of melanocytes in melasma skin, and observe the morphology and biological properties of the cultured melanocytes in vitro. To observe the effect of different concentrations of exogenous estrogen(diethylstilbestrol)on cultured human melanocyte in face. And to observe the effect of estrogen on the tyrosinase activity and melanin synthesis of the cultured human facial skin melanocytes.3,To investigate and evaluate the application of computer analysis software system on measurement of human pigmentation diseases. To investigate the ways to treat melasma with comprehensive methods, and to improve the treatment effectiveness of melasma. To investigate the melanin distribution before and after laser treatment in melasma patients in order to understand the melanin metabolism changes.Methods1,The excess skin in the lateral canthus was acquired after eyelid plasty operation for melasma patients, the paraffin sections was stained with HE, Fontana-Masson and CK10 immunohistochemical staining to observe the pathological changes of the melasma skin and the distribution of CK10. The excess skin in the lateral canthus was acquired during the blepharoplasty of melasma patients, the paraffin sections of which were stained with HE and Fontana-Masson to observe the melanin distribution in the melasma skin and normal skin.2,The pigment characteristics were analyzed in 25 cases melasma patients with angel software system, serum estrogen level was detected using radioimmunoassay. The distribution of ERβwas detected by immunohistochemical stain in the pigmented lesion and normal skin in melasma patients, then the correlation of its expression intensity and severity of pigment was analyzed for comparison. The skin specimen was obtained from melasma patients after lower blepharoplasty. Epidermis was isolated by 0.25% dispase, and then single cell suspension was obtained after digested by 0.25% trypsin and 0.02%EDTA. M254/HMGS conditioned media was added to cultured the cells purifiedly, the cell morphology was observed through inverted microscope, S-100 protein immunohistochemical staining and dopa-staining was used for identification. The TRP-1, HMB-45 expression were studied by indirect immunofluorescence. The primary melanocytes were cultured from face skin. The 3rd generation subcultured melanocytes were seeded in 96 well plates, and each hole was added for 5×103 cells. The medium was exchanged after 24h, and various concentrations of diethylstilbestrol from 1×10-4～1×10-8M was added to medium. The melanocyte proliferation was determinated by MTT method after incubation for 72h. The excess skin in the lateral canthus was acquired during the blepharoplasty. The primary melanocytes were cultured and identified. The second generation cells were passaged and inoculated. The medium was changed after 24 hours, added with medium contained estrogen. After 48 hours, tyrosinase activity and pigment synthesis were detected, and the ultrastructural changes were detected after 72 hours. 3,Digital photos of pigment disorders in patients were collected before and after the treatment, and then were input the computer. The pigment analysis software system was applied to measure the area of lesions, and the red, green, blue gray value and the average gray of the facial pigment in patients, also include of the largest and the smallest gray.600 patients with melasma were treated with topical medications, infusion therapy, oral medications and lasers, intense pulsed light (IPL) and other comprehensive methods. To assess the effectiveness before and after treatment using pigment analysis software. Melasma patients whose lower eyelid skin was loosening were chosen. A split face study was carried on. One side of the face was treated with laser firstly for 3 times. lweek-l month after the last laser treatment, bilateral lower blepharoplasty was carried out. The skin of both sides was stained by HE and Fontana-Masson respectively to observe the change of pigment granules distribution in melasma lesions before and after laser treatment.Results1,The epidermis was thin in the melasma skin. And the melanin can be seen clearly around the basal layer cells.It could be seen in the upper basal layer, the upper hair follicle and dermis. Dermal collagen was disorder, fracture, the number of skin appendages was less than the normal skin. CK10 mainly distributed in the upper stratum of basal epidermis and the most outer layer of sebaceous glands. The melanin mainly distributed in the basal layer and the rete ridge(nail-like protrusion) in the melasma lesions, which was liked umbrella or cap wrapping around the nucleus. A number of melanin also distributed in the upper section of the hair follicles, the upper stratum of the basement and the dermis. Melanin was significantly more compared with the non-pigmented skin. Less melanin could be found in non-pigmented skin, but the corneum was significantly thicker. 2,The lesion on melasma skin was mostly brown. The average gray value was 122.84±20.59, estradiol(E2),progesterone(P),luteinizing hormone(LH) and follicle stimulating hormone(FSH) in vivo is higher compared with normal control group, and testosterone(T), prolactin(PRL) have no significantly difference with normal. ERβexpressed strongly in the nucleus of the cell of epidermis and hair follicle's outer root sheath in pigmentation lesion, and weaken from basal layer of epidermis to superficial. It expressed also in sebaceous glands, sweat glands, dermis fibroblast and vessel endothelial cell. It is positive correlation between the expression ERP in the epidermis and the severity of pigment. The human melanocytes in melasma skin were successfully cultured, and the cells were multi-dendritic. S-100 protein immunohistochemical staining and Dopa staining were positive. The TRP-1 and HMB-45 were positive expression by indirect immunofluorescence.10-8-10-6 M diethylstilbestrol could promote the proliferation of cultured melanocytes, and the intensity was positively correlated with the diethylstilbestrol concentration.10-5 M or more clould inhibited the proliferation of melanocytes. The tyrosinase activity and melanin synthesis were increased significantly after 10"6M estrogen was added (P< 0.05). The melanin bodies showed an increase in melanocytes by transmission electron microscope.3,With pigment analysis computer software system, the area and the average gray value of the pigmented disease can be measured. The area of lesions reduction and the average gray value increase indicated the therapy is effective. After combined treatments, the pigmentation faded in 144 patients (24%), improved significantly in 168 patients (28%), improved mildly in 204 patients (34%), and the total effective rate was about 86%. Melasma lesions pigment mainly surrounded the basal cells. Certain of melanin distributed in the upper stratum of the basement, some located in the dermis. The pigment granules scattered and became smaller after laser treatment, some of which migrate to the upper stratum. Some of pigment granules located in the cuticle. The pigment granules in dermis also increased significantly after laser treatment.Conclusion1,The content of melanin increases in melasma skin, and skin aging features appear in the histological structure. CK10 expresses strongly in the upper stratum of basal epidermis which maybe relate with the excessive differentiation of epidermal cells and the skin aging.Melasma melanin mainly distributes in the basal layer and the rete ridge, and is significantly more compared with that in the normal skin.2,The changes of estrogen in vivo may play an important role in the pathogenesis of melasma. ERβexpresses in the skin and it's appendant. It may be playing an important role in the process of the skin aging and skin pigmentation. Melanocytes can be successfully cultured in melasma skin with conditioned media insisted of special ingredients, which can be used for further investigation. The concentration of diethylstilbestrol can promote the cultured melanocytes proliferation in melasma, and the best concentration is 10-6M. The estrogen may play a important role on the incidence of melasma. Certain concentration of estrogen can improve the activity of tyrosinase and promote synthesis of melanin in melanocytes.3,Computer pigment analysis software systems can be applied to assess the pigmented lesions objectively and scientificly. Different treatments should be selected according to the condition of patients, and a satisfactory effectiveness could be achieved with combined treatments for melasma. Laser treatment can accelerate the metabolism of melasma pigment granules.
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